PARP inhibitor compounds, compositions and methods of use

ABSTRACT

The present invention relates to tetraaza phenalen-3-one compounds which inhibit poly (ADP-ribose) polymerase (PARP) and are useful in the chemosensitization of cancer therapeutics. The induction of peripheral neuropathy is a common side-effect of many of the conventional and newer chemotherapies. The present invention further provides means to reliably prevent or cure chemotherapy-induced neuropathy. The invention also relates to the use of the disclosed PARP inhibitor compounds in enhancing the efficacy of chemotherapeutic agents such as temozolomide. The invention also relates to the use of the disclosed PARP inhibitor compounds to radiosensitize tumor cells to ionizing radiation. The invention also relates to the use of the disclosed PARP inhibitor compounds for treatment of cancers with DNA repair defects.

This application claims the benefit of U.S. Provisional Application No.60/977,115, filed Oct. 3, 2007, the entire contents of which are herebyincorporated by reference.

FIELD OF THE INVENTION

The present invention relates to tetraaza phenalen-3-one compounds whichinhibit poly (ADP-ribose) polymerase (PARP).

BACKGROUND

The present invention relates to inhibitors of the nuclear enzymepoly(adenosine 5′-diphospho-ribose) polymerase [“poly(ADP-ribose)polymerase” or “PARP”, which is also referred to as ADPRT (NAD:protein(ADP-ribosyl transferase (polymerising)) and PARS (poly(ADP-ribose)synthetase) and provides compounds and compositions containing thedisclosed compounds. Moreover, the present invention provides methods ofusing the disclosed PARP inhibitors to treat cancer.

There is considerable interest in the development of PARP inhibitors aschemosensitizers for use in cancer therapy and to limit cellular damageafter ischemia or endotoxic stress. In particular, potentiation oftemozolomide cytotoxicity observed in preclinical studies with potentPARP-1 inhibitors reflects inhibition of base excision repair andsubsequent cytotoxicity due to incomplete processing of N⁷-methylguanineand N³-methyladenine. There is now a body of preclinical datademonstrating that the cytotoxicity of temozolomide is potentiated bycoadministration of a PARP inhibitor either in vitro or in vivo.Plummer, et al., Clin. Cancer Res., 11(9), 3402 (2005).

Temozolomide, a DNA methylating agent, induces DNA damage, which isrepaired by O⁶-alkylguanine alkyltransferase (ATase) andpoly(ADP-ribose) polymerase-1 (PARP-1)-dependent base excision repair.Temozolomide is an orally available monofunctional DNA alkylating agentused to treat gliomas and malignant melanoma. Temozolomide is rapidlyabsorbed and undergoes spontaneous breakdown to form the activemonomethyl triazene, 5-(3-methyl-1-triazeno)imidazole-4-carboxamide.Monomethyl triazene forms several DNA methylation products, thepredominate species being N⁷-methylguanine (70%), N³-methyladenine (9%),and O⁶-methylguanine (5%). Unless repaired by O⁶-alkylguaninealkyltransferase, O⁶-methylguanine is cytotoxic due to mispairing withthymine during DNA replication. This mispairing is recognized on thedaughter strand by mismatch repair proteins and the thymine excised.However, unless the original O⁶-methylguanine nucleotide in the parentstrand is repaired by ATase-mediated removal of the methyl adduct,thymine can be reinserted. Repetitive futile rounds of thymine excisionand incorporation opposite an unrepaired O⁶-methylguanine nucleotidecauses a state of persistent strand breakage and the MutS branch ofmismatch repair system signals G2-M cell cycle arrest and the initiationof apoptosis. The quantitatively more important N⁷-methylguanine andN³-methyladenine nucleotide alkylation products formed by temozolorideare rapidly repaired by base excision repair. Plummer, et al., Clin.Cancer Res., 11(9), 3402 (2005).

Chemosensitization by PARP inhibitors is not limited to temozolomide.Cytotoxic drugs, generally, or radiation can induce activation ofPARP-1, and it has been demonstrated that inhibitors of PARP-1 canpotentiate the DNA damaging and cytotoxic effects of chemotherapy andirradiation. Kock, et al., 45 J Med. Chem. 4961 (2002). PARP-1 mediatedDNA repair in response to DNA damaging agents represents a mechanism fordrug resistance in tumors, and inhibition of this enzyme has been shownto enhance the activity of ionizing radiation and several cytotoxicantitumor agents, including temozolomide and topotecan. Suto et al, inU.S. Pat. No. 5,177,075, disclose several isoquinolines used forenhancing the lethal effects of ionizing radiation or chemotherapeuticagents on tumor cells. Weltin et al., “Effect of 6(5H)-Phenanthridinone,an Inhibitor of Poly(ADP-ribose) Polymerase, on Cultured Tumor Cells”,Oncol Res., 6:9, 399-403 (1994) disclose the inhibition of PARPactivity, reduced proliferation of tumor cells, and a marked synergisticeffect when tumor cells are co-treated with an alkylating drug. PARP-1is thus a potentially important therapeutic target for enhancingDNA-damaging cancer therapies.

PARP inhibitors can also inhibit the growth of cells having defects inthe homologous recombination (HR) pathway of double-stranded DNA repair.See Bryant et al., “Specific killing of BRCA2-deficient tumours withinhibitors of poly(ADP-ribose) polymerase,” Nature 434, 913 (2005);Farmer et al., “Targeting the DNA repair defect in BRCA mutant cells asa therapeutic strategy,” Nature 434, 917 (2005). This effect operateswithout the presence of chemosensitizers. Id. Known states associatedwith HR defects include BRCA-1 defects, BRCA-2 defects, and Fanconianemia-associated cancers. McCabe et al., “Deficiency in the Repair ofDNA Damage by Homologous Recombination and Sensitivity toPoly(ADP-Ribose) Polymerase Inhibition,” Cancer Res. 66. 8109 (2006).Proteins identified as associated with a Fanconi anemia include FANCA,FANCB, FANCC, FANCD2, FANCE, FANCF, FANCG, FANCL, and FANCM. Id. Forreviews, see Zaremba et al., “PARP Inhibitor Development for SystemicCancer Targeting,” Anti-Cancer Agents in Medicinal Chemistry 7, 515(2007) and Lewis et al., “Clinical poly(ADP-ribose) polymeraseinhibitors for the treatment of cancer,” Curr Opin. InvestigationalDrugs 8, 1061 (2007).

Large numbers of known PARP inhibitors have been described in Banasik etal., “Specific Inhibitors of Poly(ADP-Ribose) Synthetase andMono(ADP-Ribosyl)-Transferase”, J. Biol. Chem., 267:3, 1569-75 (1992),and in Banasik et al., “Inhibitors and Activators of ADP-RibosylationReactions”, Molec. Cell. Biochem., 138, 185-97 (1994). However,effective use of these PARP inhibitors, in the ways discussed above, hasbeen limited by the concurrent production of unwanted side-effects. SeeMilam et al., “Inhibitors of Poly(Adenosine Diphosphate-Ribose)Synthesis; Effect on Other Metabolic Processes,” Science, 223, 589-91(1984).

In addition to the above, PARP inhibitors have been disclosed anddescribed in the following international patent applications: WO00/42040; WO 00/39070; WO 00/39104; WO 99/11623; WO 99/11628; WO99/11622; WO 99/59975; WO 99/11644; WO 99/11945; WO 99/11649; and WO99/59973. A comprehensive review of the state of the art has beenpublished by Li and Zhang in IDrugs 2001, 4(7): 804-812 (PharmaPress LtdISSN 1369-7056).

The ability of PARP-inhibitors to potentiate the lethality of cytotoxicagents by chemosensitizing tumor cells to the cytotoxic effects ofchemotherapeutic agents has been reported in, inter alia, US2002/0028815; US 2003/0134843; US 2004/0067949; White A W, et al., 14Bioorg. and Med. Chem. Letts. 2433 (2004); Canon Koch S S, et al., 45 J.Med. Chem. 4961 (2002); Skalitsky D J, et al., 46 J. Med. Chem. 210(2003); Farmer H, et al., 434 Nature 917 (14 Apr. 2005); Plummer E R, etal., 11(9) Clin. Cancer Res. 3402 (2005); Tikhe J G, et al., 47 J. Med.Chem. 5467 (2004); Griffin R. J., et al., WO 98/33802; and Helleday T,et al, WO 2005/012305.

The induction of peripheral neuropathy is a common factor in limitingtherapy with chemotherapeutic drugs. Quasthoff and Hartung, J.Neurology, 249, 9-17 (2002). Chemotherapy induced neuropathy is aside-effect encountered following the use of many of the conventional(e.g., Taxol, vincritine, cisplatin) and newer chemotherapies (e.g.velcade, epothilone). Depending on the substance used, a pure sensoryand painful neuropathy (with cisplatin, oxaliplatin, carboplatin) or amixed sensorimotor neuropathy with or without involvement of theautonomic nervous system (with vincristine, taxol, suramin) can ensue.Neurotoxicity depends on the total cumulative dose and the type of drugused. In individual cases neuropathy can evolve even after a single drugapplication. The recovery from symptoms is often incomplete and a longperiod of regeneration is required to restore function. Up to now, fewdrugs are available to reliably prevent or cure chemotherapy-inducedneuropathy.

There continues to be a need for effective and potent PARP inhibitorswhich enhance the lethal effects of chemotherapeutic agents on tumorcells while producing minimal side-effects.

In addition, PARP inhibitors have been reported to be effective inradiosensitizing hypoxic tumor cells and effective in preventing tumorcells from recovering from potentially lethal damage of DNA afterradiation therapy, presumably by their ability to prevent DNA repair.U.S. Pat. Nos. 5,032,617; 5,215,738; and 5,041,653.

Recent publications suggest that PARP inhibitors kill breast cancercells that are deficient in breast cancer associated gene-1 and -2(BRCA1/2). These studies suggest that PARP inhibitors may be effectivefor treating BRCA1/2-associated breast cancers. [Farmer et al., Nature2005, 434, 917; DeSoto and Deng, Intl. J. Med. Sci. 2006, 3, 117; Bryantet al., Nature, 2005, 434, 913.]

There continues to be a need for effective and potent PARP inhibitorswhich enhance the lethal effects of ionizing radiation and/orchemotherapeutic agents on tumor cells, or inhibit the growth of cellshaving defects in the homologous recombination (HR) pathway ofdouble-stranded DNA repair, while producing minimal side-effects.

SUMMARY OF INVENTION

The present invention provides compounds described herein, derivativesthereof and their uses to inhibit poly(ADP-ribose) polymerase (“PARP”),compositions containing these compounds and methods for making and usingthese PARP inhibitors to treat the effects of the conditions describedherein.

The present invention also provides a tetraaza phenalen-3-one compoundof Formula (I), or a pharmaceutically acceptable salt thereof:

wherein R is(a) NR¹R², wherein R¹ is selected from the group consisting of hydrogen,C₁-C₆ straight or branched chain alkyl, C₂-C₆ straight or branched chainalkenyl, C₃-C₈ cycloalkyl, C₁-C₆ alkoxy, C₂-C₆ alkenyloxy, phenyl,phenoxy, benzyloxy,NR^(A)R^(B) (C₁-C₆ straight or branched chain alkyl), NR^(A)R^(B) (C₂-C₆straight or branched chain alkenyl), (C₁-C₆ straight or branched chainalkyl)carbonyl, (C₂-C₆ straight or branched chain alkenyl)carbonyl,(C₃-C₈ cycloalkyl)carbonyl,(C₁-C₆ straight or branched chain alkyl)oxycarbonyl, (C₂-C₆ straight orbranched chain alkenyl)oxycarbonyl, (C₃-C₈ cycloalkyl)oxycarbonyl,arylcarbonyl, sulfonyl, arylsulfonyl, aryl(C₁-C₆ straight or branchedchain alkyl), aryl(C₂-C₆ straight or branched chain alkenyl), aryl(C₃-C₈cycloalkyl),(C₁-C₆ straight or branched chain alkyl)aryl, (C₂-C₆ straight orbranched chain alkenyl)aryl, (C₃-C₈ cycloalkyl)aryl, aryl,heterocyclyl, heterocyclyl(C₁-C₆ straight or branched chain alkyl), andheterocyclyl(C₂-C₆ straight or branched chain alkenyl); wherein eachheterocyclyl has between 1 and 7 heteroatoms independently selected fromO, N, or S, and wherein each of R^(A) and R^(B) are independentlyselected from the group consisting of hydrogen, C₁-C₆ straight orbranched chain alkyl, C₂-C₆ straight or branched chain alkenyl, andC₃-C₈ cycloalkyl;and R² is selected from the group consisting of C₁-C₆ straight orbranched chain alkyl, C₂-C₆ straight or branched chain alkenyl, C₃-C₈cycloalkyl, C₁-C₆ alkoxy, C₂-C₆ alkenyloxy, phenyl, phenoxy, benzyloxy,NR^(X)R^(Y) (C₁-C₆ straight or branched chain alkyl), NR^(X)R^(Y) (C₂-C₆straight or branched chain alkenyl), (C₁-C₆ straight or branched chainalkyl)carbonyl, (C₂-C₆ straight or branched chain alkenyl)carbonyl,(C₃-C₈ cycloalkyl)carbonyl, (C₁-C₆ straight or branched chainalkyl)oxycarbonyl, (C₂-C₆ straight or branched chainalkenyl)oxycarbonyl,(C₃-C₈ cycloalkyl)oxycarbonyl, arylcarbonyl, sulfonyl, arylsulfonyl,aryl(C₁-C₆ straight or branched chain alkyl), aryl(C₂-C₆ straight orbranched chain alkenyl), aryl(C₃-C₈ cycloalkyl), (C₁-C₆ straight orbranched chain alkyl)aryl, (C₂-C₆ straight or branched chainalkenyl)aryl, (C₃-C₈ cycloalkyl)aryl, aryl,heterocyclyl, heterocyclyl(C₁-C₆ straight or branched chain alkyl), andheterocyclyl(C₂-C₆ straight or branched chain alkenyl); wherein eachheterocyclyl has between 1 and 7 heteroatoms independently selected fromO, N, or S, and wherein each of R^(X) and R^(Y) are independentlyselected from the group consisting of hydrogen, C₁-C₆ straight orbranched chain alkyl, C₂-C₆ straight or branched chain alkenyl, andC₃-C₈ cycloalkyl;wherein R¹ and R² are independently substituted with between 0 and 4substituents, each independently selected from halo, C₁-C₆ straight orbranched chain alkyl, C₂-C₆ straight or branched chain alkenyl, C₁-C₆alkoxy, trifluoromethyl, trifluoroethyl, and amino; and provided that R¹and R² may not both be methyl, and R² may not be (phenyl)prop-1-yl whenR¹ is hydrogen; or(b) aryloxy, substituted with between 0 and 4 substituents, eachindependently selected from the group consisting of halo, C₁-C₆ alkoxy,trifluoromethyl, trifluoroethyl, C₁-C₆ straight or branched chain alkyl,C₂-C₆ straight or branched chain alkenyl, C₃-C₈ cycloalkyl, NR^(C)R^(D),NR^(C)R^(D)(C₁-C₆ straight or branched chain alkyl), andNR^(C)R^(D)(C₂-C₆ straight or branched chain alkenyl), wherein each ofR^(C) and R^(D) is independently selected from the group consisting ofhydrogen, C₁-C₆ straight or branched chain alkyl, C₂-C₆ straight orbranched chain alkenyl, and C₃-C₈ cycloalkyl; and when more than onesubstituent is of the form NR^(C)R^(D), each occurrence of R^(C) andR^(D) is independently selected from the group consisting of hydrogen,C₁-C₆ straight or branched chain alkyl, C₂-C₆ straight or branched chainalkenyl, and C₃-C₈ cycloalkyl; or(c) a heterocyclyl having between 1 and 7 heteroatoms independentlyselected from O, N, or S; and having between 0 and 4 substituentsindependently selected from the group consisting of halo, haloalkyl,hydroxyl, nitro, trifluoromethyl, trifluoroethyl, C₁-C₆ straight orbranched chain alkyl, C₂-C₆ straight or branched chain alkenyl, C₁-C₆alkoxy, C₂-C₆ alkenyloxy, phenyl, phenoxy, benzyloxy, amino,thiocarbonyl, cyano, imino, NR^(E)R^(F)(C₁-C₆ straight or branched chainalkyl), NR^(E)R^(F)(C₂-C₆ straight or branched chain alkenyl)sulflhydryl, thioalkyl, dioxa-spiroethyl, (C₁-C₆ straight or branchedchain alkyl) carbonyl, (C₂-C₆ straight or branched chainalkenyl)carbonyl, (C₁-C₆ straight or branched chain alkyl)oxycarbonyl,(C₂-C₆ straight or branched chain alkenyl)oxycarbonyl, arylcarbonyl,sulfonyl, arylsulfonyl, aryl(C₁-C₆ straight or branched chain alkyl),aryl(C₂-C₆ straight or branched chain alkenyl), aryl(C₃-C₈ cycloalkyl),(C₁-C₆ straight or branched chain alkyl)aryl, (C₂-C₆ straight orbranched chain alkenyl)aryl, (C₃-C₈ cycloalkyl)aryl, aryl, heterocyclyl,heterocyclyl(C₁-C₆ straight or branched chain alkyl), andheterocyclyl(C₂-C₆ straight or branched chain alkenyl), wherein eachheterocyclyl has between 1 and 7 heteroatoms independently selected fromO, N, or S, wherein each of R^(E) and R^(F) is independently selectedfrom the group consisting of hydrogen, C₁-C₆ straight or branched chainalkyl, C₂-C₆ straight or branched chain alkenyl, and C₃-C₈ cycloalkyl;and when more than one substituent is of the form NR^(E)R^(F) eachoccurrence of R^(E) and R^(F) is independently selected from the groupconsisting of hydrogen, C₁-C₆ straight or branched chain alkyl, C₂-C₆straight or branched chain alkenyl, and C₃-C₈ cycloalkyl; wherein eachof said 0-4 substituents is independently substituted with between 0 and4 further substituents, and each said further substituent isindependently selected from halo, C₁-C₆ straight or branched chainalkyl, C₂-C₆ straight or branched chain alkenyl, C₃-C₈ cycloalkyl, C₁-C₆alkoxy, trifluoromethyl, trifluoroethyl, and amino; provided that R hasat least one substituent when R is an N-piperidinyl, N-pyrrolidinyl oran N-morpholinyl group.

In some embodiments each ring of each heterocyclyl of Formula (I) isindependently 5-7 atoms in size.

Some embodiments include one, two or three nitrogen atoms in at leastone ring of the heterocyclyl of Formula (I).

In some embodiments, the heterocyclyl of Formula (I) comprises 1-3rings. In some embodiments, the heterocyclyl has 1-7 heteroatomsindependently selected from O, N, and S. In some embodiments, theheterocyclyl comprises 1-2 rings. In some embodiments, the heterocyclylcomprises one ring. In some embodiments, the various occurrences of theheterocyclyl of Formula (I) each independently comprise 1-3 rings. Insome embodiments, the various occurrences of the heterocyclyl of Formula(I) each independently comprise 1-2 rings. In some embodiments, thevarious occurrences of the heterocyclyl of Formula (I) eachindependently comprise one ring.

In some embodiments, the heterocyclyl of Formula (I) is selected fromthe group consisting of piperidinyl, piperazinyl, pyridazinyl,dihydropyridyl, tetrahydropyridyl, pyridinyl, pyrimidinyl,dihydropyrimidinyl, tetrahydrophyrimidinyl, hexahydropyrimidinyl,dihydropyrazinyl, tetrahydropyrazinyl, pyrrolidinyl, imidazolidinyl,pyrazolidinyl, pyrrolyl, dihydropyrolyl, imidazolyl, dihydroimidazoyl,pyrazolyl, dihydropyrazolyl, azepanyl, [1,2]diazepanyl, [1,3]diazepanyl,[1,4]diazepanyl, indolyl, dihydroindolyl, isoindolyl, dihydroisoindoly,dihydroquinolyl, tetrahydroquinolyl, dihydroisoquinolyl, andtetrahydroisoquinolyl; or subsets thereof.

The present invention also relates to a pharmaceutical compositioncomprising (i) a therapeutically amount of a compound of Formula (I) and(ii) a pharmaceutically acceptable carrier.

The present invention provides compounds which inhibit the in vitroand/or in vivo polymerase activity of poly(ADP-ribose) polymerase(PARP), and compositions containing the disclosed compounds.

The present invention provides methods to inhibit, limit and/or controlthe in vitro and/or in vivo polymerase activity of poly(ADP-ribose)polymerase (PARP) in solutions cells, tissues, organs or organ systems.In one embodiment, the present invention provides methods of limiting orinhibiting PARP activity in a mammal, such as a human, either locally orsystemically.

In one embodiment, the invention provides a chemosensitization methodfor treating cancer comprising contacting the cancer cells with acytotoxicity-potentiating tetraaza phenalen-3-one compound of Formula(I) or a pharmaceutically acceptable salt thereof and further contactingthe tumor or cancer cells with an anticancer agent.

An embodiment of the present invention provides a chemosensitizationmethod wherein a first dose of at least one compound of Formula (I) or apharmaceutically acceptable salt thereof is administered singly orrepeatedly to a patient in need thereof, and wherein subsequently asecond dose of at least one chemotherapeutic agent is administeredsingly or repeatedly to said patient after a time period to provide aneffective amount of chemosensitization.

An aspect of the present invention provides a pharmaceutical formulationcomprising the compound of Formula (I) in a form selected from the groupconsisting of Non-limiting examples of such chemotherapeutic agents arerecited below. pharmaceutically acceptable free bases, salts, hydrates,esters, solvates, stereoisomers, and mixtures thereof. According to afurther aspect, the pharmaceutical formulation further comprises apharmaceutically acceptable carrier and, optionally, a chemotherapeuticagent. The following embodiments are for illustrative purposes only andare not intended to limit in any way the scope of the present invention.In one embodiment, a pharmaceutical formulation of the inventioncomprises a compound of the invention in a pharmaceutically acceptablecarrier. In another embodiment, a pharmaceutical formulation of theinvention comprises a pharmaceutically acceptable salt of a compound ofthe invention in a pharmaceutically acceptable carrier. In anotherembodiment, a pharmaceutical formulation of the invention comprises acompound of the invention and one or more chemotherapeutic agents in apharmaceutically acceptable carrier. In another embodiment, apharmaceutical formulation of the invention comprises a pharmaceuticallyacceptable salt of a compound of the invention and one or morechemotherapeutic agents in a pharmaceutically acceptable carrier.Non-limiting examples of such chemotherapeutic agents are recited below.

According to additional aspects of the invention, the chemosensitizingcompound and the chemotherapeutic agent are administered essentiallysimultaneously.

According to an aspect of the invention, the chemotherapeutic agent isselected from the group consisting of temozolomide, adriamycin,camptothecin, carboplatin, cisplatin, daunorubicin, docetaxel,doxorubicin, interferon-alpha, interferon-beta, interferon-gamma,interleukin 2, irinotecan, paclitaxel, a taxoid, dactinomycin,danorubicin, 4′-deoxydoxorubicin, bleomycin, pilcamycin, mitomycin,neomycin and gentamycin, etoposide, 4-OH cyclophosphamide, a platinumcoordination complex, topotecan, therapeutically effective analogs andderivatives of the same, and mixtures thereof. According to a specificaspect, the chemotherapeutic agent is temozolomide.

In another embodiment, the present invention provides methods oftreating the effect of cancer and/or to radiosensitize cancer cells torender the cancer cells more susceptible to radiation therapy andthereby to prevent the tumor cells from recovering from potentiallylethal damage of DNA after radiation therapy, comprising administeringto a subject an effective amount of a compound of Formula (I) or apharmaceutically acceptable salt thereof A method of this embodiment isdirected to specifically and preferentially radiosensitizing cancercells rendering the cancer cells more susceptible to radiation therapythan non-tumor cells.

The present invention also provides a method of treatment of cancer in asubject in need thereof comprising administering to the subject atherapeutically effective amount of a compound of Formula (I) or apharmaceutically acceptable salt thereof, wherein the cancer cells havea defect in repair of double-stranded DNA scission. In one embodiment,the defect in repair of double-stranded DNA scission is a defect inhomologous recombination. In one embodiment, the cancer cells have aphenotype selected from the group consisting of a BRCA-1 defect, aBRCA-2 defect, a BRCA-1 and BRCA-2 defect, and Fanconi anemia.

In another embodiment, the present invention provides methods oftreating BRCA1/2-associated breast cancer comprising administering acompound of Formula (I) or a pharmaceutically acceptable salt thereof.

According to one embodiment of the invention, the compound for use inthe chemosensitization method of the invention, the radiosensitizationmethod of the invention, or the treatment of cancer wherein the cancercells have a defect in repair of double-stranded DNA scission method ofthe invention, is a compound selected from Formula (I) or apharmaceutically acceptable salt thereof. In another aspect, thecompound is selected from the group consisting of

and pharmaceutically acceptable salts thereof.

The present invention also provides means to treat chemotherapy-inducedperipheral neuropathy. According to an aspect of the invention, thecompounds of the present invention are administered prior to, ortogether with, the administration of at least one chemotherapy agent toprevent the development of neuropathy symptoms or to mitigate theseverity of such symptoms. According to a further aspect, the compoundsof the present invention are administered after the administration of atleast one chemotherapeutic agent to treat a patient for the symptoms ofneuropathy or to mitigate the severity of such symptoms. In anotheraspect, the present invention provides a method to retard, delay, orarrest the growth of cancer cells in a mammal, comprising theadministration of a chemotherapeutic agent, and further comprising theadministration of a compound of Formula (I) or a pharmaceuticallyacceptable salt thereof in an amount sufficient to potentiate theanticancer activity of said chemotherapeutic agent.

Still other aspects and advantages of the present invention will becomereadily apparent by those skilled in the art from the following detaileddescription, wherein it is shown and described preferred embodiments ofthe invention, simply by way of illustration of the best modecontemplated of carrying out the invention. As will be realized theinvention is capable of other and different embodiments, and its severaldetails are capable of modifications in various obvious respects,without departing from the invention. Accordingly, the description is tobe regarded as illustrative in nature and not as restrictive.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1.—The oral administration of PARP-1 inhibitor Compound 13+TMZdemonstrating the enhance survival of mice bearing the B16 melanomamodel.

FIG. 2.—The oral administration of PARP-1 inhibitor Compound 13+TMZdemonstrating the enhanced survival in the intracranial SJGBM gliomamodel.

FIG. 3.—The oral administration of PARP-1 inhibitor Compound 37+TMZdemonstrating the enhance survival of mice bearing the B16 melanomamodel.

FIG. 4.—The oral administration of PARP-1 inhibitor Compound 37+TMZdemonstrating the enhanced survival in the intracranial SJGBM gliomamodel.

FIG. 5.—The oral administration of PARP-1 inhibitor Compound37+radiation demonstrating inhibition of tumor growth in the model ofhead and neck cancer.

FIG. 6.—The oral administration of PARP-1 inhibitor Compound 37demonstrating inhibition of growth of BRCA1 mutant tumors

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides compounds described herein, derivativesthereof and their uses to inhibit poly(ADP-ribose) polymerase (“PARP”),compositions containing these compounds and methods for making and usingthese compounds to treat, prevent and/or ameliorate the effects ofcancers by potentiating the cytotoxic effects of ionizing radiation ontumor cells.

The present invention provides compounds described herein, derivativesthereof and their uses to inhibit poly(ADP-ribose) polymerase (“PARP”),compositions containing these compounds and methods for making and usingthese compounds to treat the effects of cancers by potentiating thecytotoxic effects of chemotherapeutic agents on tumor cells.

The present invention provides a chemosensitization method for treatingtumor and/or cancer cells comprising contacting said cancer cells with acompound of Formula (I) and further contacting said cancer cells with ananticancer agent.

The present invention provides compounds described herein, derivativesthereof and their uses to inhibit poly(ADP-ribose) polymerase (“PARP”),compositions containing these compounds and methods for making and usingthese compounds to inhibit the growth of cells having defects in thehomologous recombination (HR) pathway of double-stranded DNA repair.

The compounds and compositions of the present invention can be used inthe presence or absence of radio- or chemo-sensitizers for the treatmentof cancer. The compounds and compositions are preferably used in theabsence of radio- or chemo-sensitizers where the cancer has a defect inthe homologous recombination (HR) pathway of double-stranded DNA repair.Such defects are associated with, and have the phenotypes of, BRCA-1defects, BRCA-2 defects, dual BRCA-1/BRCA-2 defects, and Fanconi anemia.

Fanconi anemia is a genetically heterogeneous disease and patients withFanconi anemia have a greatly increased risk of cancer. Eleven proteinshave been associated with Fanconi anemia. FANCA, FANCB, FANCC, FANCE,FANCF, FANCG, and FANCM form a nuclear core complex. The complexinteracts with FANCL to incorporate ubiquinone of FANCD2. ModifiedFANCD2 is need for repair of DNA cross-links. FANCd2 accumulates atsites of DNA damage and associates with BRCA-1 and BRCA-2.

Exemplary cancers that can be associated with HR defects include breastcancer and ovarian cancer. Breast cancer for treatment by the methods ofthe invention can include all types of breast cancer and preferablyincludes invasive ductal carcinoma and invasive lobular carcinoma.Ovarian cancer for treatment by the methods of the invention include alltypes of ovarian cancer, preferably epithelial ovarian tumors, germ cellovarian tumors, and sex cord stromal tumors.

The compounds of the present invention can be synthesized using thestarting materials and methods disclosed in U.S. application Ser. No.10/853,714, which is incorporated herein by reference in its entirety.

Typically, the compounds, such as those of Formula (I), used in thecompositions of the invention will have an IC₅₀ for inhibitingpoly(ADP-ribose) polymerase in vitro of about 20 μM or less, preferablyless than about 10 μM, more preferably less than about 1 μM, orpreferably less than about 0.1 μM, most preferably less than about 0.01μM.

A convenient method to determine IC₅₀ of a PARP inhibitor compound is aPARP assay using purified recombinant human PARP from Trevigan(Gaithersburg, Md.), as follows: The PARP enzyme assay is set up on icein a volume of 100 microliters consisting of 100 mM Tris-HCl (pH 8.0), 1mM MgCl₂, 28 mM KCl, 28 mM NaCl, 3.0 μg/ml of DNase I-activated herringsperm DNA (Sigma, Mo.), 30 micromolar [³H]nicotinamide adeninedinucleotide (62.5 mCi/mmole), 15 micrograms/ml PARP enzyme, and variousconcentrations of the compounds to be tested. The reaction is initiatedby adding enzyme and incubating the mixture at 25° C. After 2 minutes ofincubation, the reaction is terminated by adding 500 microliters of icecold 30% (w/v) trichloroacetic acid. The precipitate formed istransferred onto a glass fiber filter (Packard Unifilter-GF/C) andwashed three times with 70% ethanol. After the filter is dried, theradioactivity is determined by scintillation counting. The compounds ofthis invention were found to have potent enzymatic activity in the rangeof a few nanomolar to 20 micromolar in IC₅₀ in this inhibition assay.

As used herein, “alkyl” means a branched or unbranched saturatedhydrocarbon chain comprising a designated number of carbon atoms. Forexample, C₁-C₆ straight or branched alkyl hydrocarbon chain contains 1to 6 carbon atoms, and includes but is not limited to substituents suchas methyl, ethyl, propyl, iso-propyl, butyl, iso-butyl, tert-butyl,n-pentyl, n-hexyl, and the like, unless otherwise indicated. In someembodiments, the alkyl chain is a C₁ to C₆ branched or unbranched carbonchain. In some embodiments, the alkyl chain is a C₂ to C₅ branched orunbranched carbon chain. In some embodiments, the alkyl chain is a C₁ toC₄ branched or unbranched carbon chain. In some embodiments, the alkylchain is a C₂ to C₄ branched or unbranched carbon chain. In someembodiments, the alkyl chain is a C₃ to C₅ branched or unbranched carbonchain. In some embodiments, the alkyl chain is a C₁ to C₂ branched orunbranched carbon chain. In some embodiments, the alkyl chain is a C₂ toC₃ branched or unbranched carbon chain.

“Alkenyl” means a branched or unbranched unsaturated hydrocarbon chaincomprising a designated number of carbon atoms. For example, C₂-C₆straight or branched alkenyl hydrocarbon chain contains 2 to 6 carbonatoms having at least one double bond, and includes but is not limitedto substituents such as ethenyl, propenyl, isopropenyl, butenyl,iso-butenyl, tert-butenyl, n-pentenyl, n-hexenyl, and the like, unlessotherwise indicated. In some embodiments, the alkenyl chain is a C₂ toC₆ branched or unbranched carbon chain. In some embodiments, the alkenylchain is a C₂ to C₅ branched or unbranched carbon chain. In someembodiments, the alkenyl chain is a C₂ to C₄ branched or unbranchedcarbon chain. In some embodiments, the alkenyl chain is a C₃ to C₅branched or unbranched carbon chain.

“Alkoxy”, means the group —OZ wherein Z is alkyl as herein defined. Zcan also be a branched or unbranched saturated hydrocarbon chaincontaining 1 to 6 carbon atoms.

“Cyclo”, used herein as a prefix, refers to a structure characterized bya closed ring.

“Halo” means at least one fluoro, chloro, bromo, or iodo moiety, unlessotherwise indicated.

Each of “NR^(A)R^(B)”, “NR^(X)R^(Y)”, “NR^(C)R^(D)”, and “NR^(E)R^(F)”as described herein independently encompass amino (NH₂) as well assubstituted amino. For example, NR^(A)R^(B) may be —NH(CH₃),—NH(cyclohexyl), and —N(CH₂CH₃)(CH₃). When more than one substituent isof the form “NR^(A)R^(B)”, “NR^(X)R^(Y)”, “NR^(C)R^(D)”, or“NR^(E)R^(F)”, each occurrence of R^(A), R^(B), R^(C), R^(D), R^(X), orR^(Y) is independently selected from the group consisting of hydrogen,C₁-C₆ straight or branched chain alkyl, C₂-C₆ straight or branched chainalkenyl, and C₃-C₈ cycloalkyl. Such examples are for illustrativepurposes only and are not intended to be limiting in any way.

“Arylcarbonyl” refers to a carbonyl radical substituted with aryl asdescribed herein. Non-limiting examples include phenylcarbonyl andnaphthylcarbonyl.

“Alkylcarbonyl” refers to a carbonyl radical substituted with alkyl asdescribed herein. Non-limiting examples include acyl and propylcarbonyl.

“Alkoxycarbonyl” refers to a carbonyl radical substituted with alkoxy asdescribed herein. Non-limiting examples include methoxycarbonyl andtert-butyloxycarbonyl.

“Ar” or “aryl” refer to an aromatic carbocyclic moiety having one ormore closed rings. Examples include, without limitation, phenyl,naphthyl, anthracenyl, phenanthracenyl, biphenyl, and pyrenyl.

“Heterocyclyl” refers to a cyclic moiety having one or more closedrings, with one or more heteroatoms (for example, oxygen, nitrogen orsulfur) in at least one of the rings, and wherein the ring or rings mayindependently be aromatic, nonaromatic, fused, and/or bridged, Examplesinclude without limation piperidinyl, piperazinyl, pyridazinyl,dihydropyridyl, tetrahydropyridyl, pyridinyl, pyrimidinyl,dihydropyrimidinyl, tetrahydrophyrimidinyl, hexahydropyrimidinyl,dihydropyrazinyl, tetrahydropyrazinyl, pyrrolidinyl, imidazolidinyl,pyrazolidinyl, pyrrolyl, dihydropyrolyl, imidazolyl, dihydroimidazoyl,pyrazolyl, dihydropyrazolyl, azepanyl, [1,2]diazepanyl, [1,3]diazepanyl,[1,4]diazepanyl, indolyl, dihydroindolyl, isoindolyl, dihydroisoindoly,dihydroquinolyl, tetrahydroquinolyl, dihydroisoquinolyl, andtetrahydroisoquinolyl.

“Arylalkyl” refers to an alkyl radical substituted with aryl.Non-limiting examples include benzyl, phenylethyl, and phenylpropyl.

“Alkylaryl” refers to an aryl radical substituted with alkyl.Non-limiting examples include tolyl and dimethylphenyl.

“Cycloalkyl” refers to a hydrocarbon cyclic moiety that is nonaromatic.Examples include without limitation cyclopropane, cyclobutane,cyclopentane, cyclohexane, cyclopheptane, cyclooctane, cyclopentene,cyclohexene, cycloheptene, and cyclooctene.

The term “nervous insult” refers to any damage to nervous tissue and anydisability or death resulting therefrom The cause of nervous insult maybe metabolic, toxic, neurotoxic, iatrogenic, thermal or chemical, andincludes without limitation, ischemia, hypoxia, cerebrovascularaccident, trauma, surgery, pressure, mass effect, hemorrhage, radiation,vasospasm, neurodegenerative disease, infection, Parkinson's disease,amyotrophic lateral sclerosis (ALS), myelination/demyelination process,epilepsy, cognitive disorder, glutamate abnormality and secondaryeffects thereof.

The term “neuroprotective” refers to the effect of reducing, arrestingor ameliorating nervous insult, and protecting, resuscitating, orreviving nervous tissue that has suffered nervous insult.

The term “preventing neurodegeneration” includes the ability to preventa neurodegenerative disease or preventing further neurodegeneration inpatients who are already suffering from or have symptoms of aneurodegenerative disease.

The term “treating” refers to:

(i) preventing a disease, disorder or condition from occurring in ananimal that may be predisposed to the disease, disorder and/orcondition, but has not yet been diagnosed as having it; and/or

(ii) inhibiting the disease, disorder or condition, i.e., arresting itsdevelopment; and/or

(iii) relieving the disease, disorder or condition, i.e., causingregression of the disease, disorder and/or condition.

The term “chemosensitizer”, as used herein, is defined as a molecule,such as a low molecular weight molecule, administered to animals intherapeutically effective amounts to potentiate the antitumoral activityof chemotherapeutic agents. Such chemosensitizers are useful, forexample, to increase the tumor growth-retarding or -arresting effect ofa given dose of a chemotherapeutic agent, or to improve the side-effectprofile of a chemotherapeutic agent by allowing for reductions in itsdose while maintaining its antitumoral efficacy.

The term “radiosensitizer”, as used herein is defined as a molecule,such as a low molecular weight molecule, administered to animals intherapeutically effective amounts to increase the sensitivity of thecells to be radiosensitized to electromagnetic radiation and/or topromote the treatment of diseases which are treatable withelectromagnetic radiation. Diseases which are treatable withelectromagnetic radiation include neoplastic diseases, benign andmalignant tumors, and cancerous cells. Electromagnetic radiationtreatment of other diseases not listed herein is also contemplated.

“Effective amount” refers to the amount required to produce the desiredeffect.

“Substituted” means that at least one hydrogen on a designated group isreplaced with another radical, provided that the designated group'snormal valence is not exceeded. With respect to any group containing oneor more substituents, such groups are not intended to introduce anysubstitution that is sterically impractical, synthetically non-feasibleand/or inherently unstable. In some embodiments of the invention asdescribed herein, a substituent may substitute a radical, which saidradical is itself a substituent. For example, in the compound shownbelow for illustrative purposes only, the piperazinyl ring is aheterocyclyl, which may be substituted with 0-4 substituents asdescribed herein. In the example compound, the piperazinyl ring issubstituted with arylsulfonyl wherein aryl is phenyl, and wherein thearylsulfonyl may be further substituted 0-4 times as described herein.In the example compound, the phenylsulfonyl moiety is furthersubstituted with tert-butyl. Such example is given for illustrativepurposes only and is not intended to be limiting in any way.

“Subject” refers to a cell or tissue, in vitro or in vivo, an animal ora human. An animal or human subject may also be referred to as a“patient.”

“Animal” refers to a living organism having sensation and the power ofvoluntary movement, and which requires for its existence oxygen andorganic food. Examples include, without limitation, members of thehuman, mammalian and primate species.

Broadly, the compounds and compositions of the present invention can beused to treat or prevent cell damage or death due to necrosis orapoptosis, cerebral ischemia and reperfusion injury or neurodegenerativediseases in an animal, such as a human. The compounds and compositionsof the present invention can be used to extend the lifespan andproliferative capacity of cells and thus can be used to treat or preventdiseases associated therewith; they alter gene expression of senescentcells; and they radiosensitize hypoxic tumor cells. Preferably, thecompounds and compositions of the invention can be used to treat orprevent tissue damage resulting from cell damage or death due tonecrosis or apoptosis, and/or effect neuronal activity, either mediatedor not mediated by NMDA toxicity. The compounds of the present inventionare not limited to being useful in treating glutamate mediatedneurotoxicity and/or NO-mediated biological pathways. Further, thecompounds of the invention can be used to treat or prevent other tissuedamage related to PARP activation, as described herein.

The present invention provides compounds which inhibit the in vitroand/or in vivo polymerase activity of poly(ADP-ribose) polymerase(PARP), and compositions containing the disclosed compounds.

The present invention provides methods to inhibit, limit and/or controlthe in vitro and/or in vivo polymerase activity of poly(ADP-ribose)polymerase (PARP) in any of solutions, cells, tissues, organs or organsystems. In one embodiment, the present invention provides methods oflimiting or inhibiting PARP activity in a mammal, such as a human,either locally or systemically.

The compounds of the invention act as PARP inhibitors to treat orprevent cancers by chemopotentiating the cytotoxic effects of thechemotherapeutic agents. The compounds of the invention act as PARPinhibitors to treat or prevent cancers by sensitizing cells to thecytotoxic effects of radiation. The compounds of the invention act asPARP inhibitors to treat or prevent BRCA1/2-associated breast cancer.

The compounds of the present invention may possess one or moreasymmetric center(s) and thus can be produced as mixtures (racemic andnon-racemic) of stereoisomers, or as individual enantiomers ordiastereomers. The individual stereoisomers may be obtained by using anoptically active staring material, by resolving a racemic or non-racemicmixture of an intermediate at some appropriate stage of the synthesis,or by resolution of the compound of Formula (I). It is understood thatthe individual stereoisomers as well as mixtures (racemic andnon-racemic) of stereoisomers are encompassed by the scope of thepresent invention.

The compounds of the invention are useful in a free base form, in theform of pharmaceutically acceptable salts, pharmaceutically acceptablehydrates, pharmaceutically acceptable esters, pharmaceuticallyacceptable solvates, pharmaceutically acceptable prodrugs,pharmaceutically acceptable metabolites, and in the form ofpharmaceutically acceptable stereoisomers. These forms are all withinthe scope of the disclosure.

“Pharmaceutically acceptable salt”, “hydrate”, “ester” or “solvate”refers to a salt, hydrate, ester, or solvate of the inventive compoundswhich possesses the desired pharmacological activity and which isneither biologically nor otherwise undesirable. Organic acids can beused to produce salts, hydrates, esters, or solvates such as acetate,adipate, alginate, aspartate, benzoate, benzenesulfonate,p-toluenesulfonate, bisulfate, sulfamate, sulfate, naphthylate,butyrate, citrate, camphorate, camphorsulfonate,cyclopentane-propionate, digluconate, dodecylsulfate, ethanesulfonate,fumarate, glucoheptanoate, glycerophosphate, hemisulfate heptanoate,hexanoate, 2-hydroxyethanesulfonate, lactate, maleate, methanesulfonate,2-naphthalenesulfonate, nicotinate, oxalate, tosylate and undecanoate.Inorganic acids can be used to produce salts, hydrates, esters, orsolvates such as hydrochloride, hydrobromide, hydroiodide, andthiocyanate.

Examples of suitable base salts, hydrates, esters, or solvates includehydroxides, carbonates, and bicarbonates of ammonia, alkali metal saltssuch as sodium, lithium and potassium salts, alkaline earth metal saltssuch as calcium and magnesium salts, aluminum salts, and zinc salts.

Salts, hydrates, esters, or solvates may also be formed with organicbases. Organic bases suitable for the formation of pharmaceuticallyacceptable base addition salts, hydrates, esters, or solvates of thecompounds of the present invention include those that are non-toxic andstrong enough to form such salts, hydrates, esters, or solvates. Forpurposes of illustration, the class of such organic bases may includemono-, di-, and trialkylamines, such as methylamine, dimethylamine,triethylamine and dicyclohexylamine; mono-, di- ortrihydroxyalkylamines, such as mono-, di-, and triethanolamine; aminoacids, such as arginine and lysine; guanidine; N-methyl-glucosamine;N-methyl-glucamine; L-glutamine; N-methyl-piperazine; morpholine;ethylenediamine; N-benzyl-phenethylamine;(trihydroxy-methyl)aminoethane; and the like. See, for example,“Pharmaceutical Salts,” J. Pharm. Sci., 66:1, 1-19 (1977). Accordingly,basic nitrogen-containing groups can be quaternized with agentsincluding: lower alkyl halides such as methyl, ethyl, propyl, and butylchlorides, bromides and iodides; dialkyl sulfates such as dimethyl,diethyl, dibutyl and diamyl sulfates; long chain halides such as decyl,lauryl, myristyl and stearyl chlorides, bromides and iodides; andaralkyl halides such as benzyl and phenethyl bromides.

The acid addition salts, hydrates, esters, or solvates of the basiccompounds may be prepared either by dissolving the free base of acompound of the present invention in an aqueous or an aqueous alcoholsolution or other suitable solvent containing the appropriate acid orbase, and isolating the salt by evaporating the solution. Alternatively,the free base of a compound of the present invention can be reacted withan acid, as well as reacting a compound of the present invention havingan acid group thereon with a base, such that the reactions are in anorganic solvent, in which case the salt separates directly or can beobtained by concentrating the solution.

“Pharmaceutically acceptable prodrug” refers to a derivative of theinventive compounds which undergoes biotransformation prior toexhibiting its pharmacological effect(s). The prodrug is formulated withthe objective(s) of improved chemical stability, improved patientacceptance and compliance, improved bioavailability, prolonged durationof action, improved organ selectivity, improved formulation (e.g.,increased hydrosolubility), and/or decreased side effects (e.g.,toxicity). The prodrug can be readily prepared from the inventivecompounds using methods known in the art, such as those described byBurgers Medicinal Chemistry and Drug Chemistry, Fifth Ed, Vol. 1, pp.172-178, 949-982 (1995). For example, the inventive compounds can betransformed into prodrugs by converting one or more of the hydroxy orcarboxy groups into esters.

“Pharmaceutically acceptable metabolite” refers to drugs that haveundergone a metabolic transformation. After entry into the body, mostdrugs are substrates for chemical reactions that may change theirphysical properties and biologic effects. These metabolic conversions,which usually affect the polarity of the compound, alter the way inwhich drugs are distributed in and excreted from the body. However, insome cases, metabolism of a drug is required for therapeutic effect. Forexample, anticancer drugs of the antimetabolite class must be convertedto their active forms after they have been transported into a cancercell. Since most drugs undergo metabolic transformation of some kind,the biochemical reactions that play a role in drug metabolism may benumerous and diverse. The main site of drug metabolism is the liver,although other tissues may also participate.

Further still, the methods of the invention can be used to treat cancerand to chemosensitize and radiosensitize cancer and/or tumor cells. Theterm “cancer,” as used herein, is defined broadly. The compounds of thepresent invention can potentiate the effects of “anti-cancer agents,”which term also encompasses “anti-tumor cell growth agents,”“chemotherapeutic agents,” “cytostatic agents,” “cytotoxic agents,” and“anti-neoplastic agents”. The term “BRCA1/2-associated breast cancer”encompasses breast cancer in which the breast cancer cells are deficientin the breast cancer tumor suppressor genes BRCA1 and/or BRCA2.

For example, the methods of the invention are useful for treatingcancers such as ACTH-producing tumors, acute lymphocytic leukemia, acutenonlymphocytic leukemia, cancer of the adrenal cortex, bladder cancer,brain cancer, breast cancer, cervical cancer, chronic lymphocyticleukemia, chronic myelocytic leukemia, colorectal cancer, cutaneousT-cell lymphoma, endometrial cancer, esophageal cancer, Ewing's sarcoma,gallbladder cancer, hairy cell leukemia, head and neck cancer, Hodgkin'slymphoma, Kaposi's sarcoma, kidney cancer, liver cancer, lung cancer(small and/or non-small cell), malignant peritoneal effusion, malignantpleural effusion, melanoma, mesothelioma, multiple myeloma,neuroblastoma, non-Hodgkin's lymphoma, osteosarcoma, ovarian cancer,ovary (germ cell) cancer, prostate cancer, pancreatic cancer, penilecancer, retinoblastoma, skin cancer, soft-tissue sarcoma, squamous cellcarcinomas, stomach cancer, testicular cancer, thyroid cancer,trophoblastic neoplasms, uterine cancer, vaginal cancer, cancer of thevulva and Wilm's tumor.

In some non-limiting embodiments, the cancer and/or tumor cells areselected from the group consisting of brain cancer, melanoma, head andneck cancer, non small cell lung cancer, testicular cancer, ovariancancer, colon cancer and rectal cancer.

The present invention also relates to a pharmaceutical compositioncomprising (i) a therapeutically effective amount of a compound of acompound of Formula (I) and (ii) a pharmaceutically acceptable carrier.

The above discussion relating to the preferred embodiments' utility andadministration of the compounds of the present invention also applies tothe pharmaceutical composition of the present invention.

The term “pharmaceutically acceptable carrier” as used herein refers toany carrier, diluent, excipient, suspending agent, lubricating agent,adjuvant, vehicle, delivery system emulsifier, disintegrant, absorbent,preservative, surfactant, colorant, flavorant, or sweetener.

For these purposes, the composition of the invention may be administeredorally, parenterally, by inhalation spray, adsorption, absorption,topically, rectally, nasally, bucally, vaginally, intraventricularly,via an implanted reservoir in dosage formulations containingconventional non-toxic pharmaceutically-acceptable carriers, or by anyother convenient dosage form The term parenteral as used herein includessubcutaneous, intravenous, intramuscular, intraperitoneal, intrathecalintraventricular, intrasternal, and intracranial injection or infusiontechniques.

When administered parenterally, the composition will normally be in aunit dosage, sterile injectable form (solution, suspension or emulsion)which is preferably isotonic with the blood of the recipient with apharmaceutically acceptable carrier. Examples of such sterile injectableforms are sterile injectable aqueous or oleaginous suspensions. Thesesuspensions may be formulated according to techniques known in the artusing suitable dispersing or wetting agents and suspending agents. Thesterile injectable forms may also be sterile injectable solutions orsuspensions in non-toxic parenterally-acceptable diluents or solvents,for example, as solutions in 1,3-butanediol. Among the acceptablevehicles and solvents that may be employed are water, saline, Ringer'ssolution, dextrose solution, isotonic sodium chloride solution, andHanks' solution. In addition, sterile, fixed oils are conventionallyemployed as solvents or suspending mediums. For this purpose, any blandfixed oil may be employed including synthetic mono- or di-glycerides,corn, cottonseed, peanut, and sesame oil. Fatty acids such as ethyloleate, isopropyl myristate, and oleic acid and its glyceridederivatives, including olive oil and castor oil, especially in theirpolyoxyethylated versions, are useful in the preparation of injectables.These oil solutions or suspensions may also contain long-chain alcoholdiluents or dispersants.

Sterile saline is a preferred carrier, and the compounds are oftensufficiently water soluble to be made up as a solution. The carrier maycontain minor amounts of additives, such as substances that enhancesolubility, isotonicity, and chemical stability, e.g., anti-oxidants,buffers and preservatives.

Formulations suitable for nasal or buccal administration (such asself-propelling powder dispensing formulations) may comprise about 0.1%to about 5% w/w, for example 1% w/w of active ingredient. Theformulations for human medical use of the present invention comprise anactive ingredient in association with a pharmaceutically acceptablecarrier therefore and optionally other therapeutic ingredient(s).

When administered orally, the composition will usually be formulatedinto unit dosage forms such as tablets, cachets, powder, granules,beads, chewable lozenges, capsules, liquids, aqueous suspensions orsolutions, or similar dosage forms, using conventional equipment andtechniques known in the art. Such formulations typically include asolid, semisolid, or liquid carrier. Exemplary carriers include lactose,dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calciumphosphate, mineral oil, cocoa butter, oil of theobroma, alginates,tragacanth, gelatin, syrup, methyl cellulose, polyoxyethylene sorbitanmonolaurate, methyl hydroxybenzoate, propyl hydroxybenzoate, talc,magnesium stearate, and the like.

The composition of the invention is preferably administered as a capsuleor tablet containing a single or divided dose of the compound of Formula(I) or pharmaceutically acceptable salt thereof. The composition may beadministered as a sterile solution, suspension, or emulsion, in a singleor divided dose. Tablets may contain carriers such as lactose and cornstarch, and/or lubricating agents such as magnesium stearate. Capsulesmay contain diluents including lactose and dried corn starch.

A tablet may be made by compressing or molding the active ingredientoptionally with one or more accessory ingredients. Compressed tabletsmay be prepared by compressing, in a suitable machine, the activeingredient in a free-flowing form such as a powder or granules,optionally mixed with a binder, lubricant, inert diluent, surfaceactive, or dispersing agent Molded tablets may be made by molding in asuitable machine, a mixture of the powdered active ingredient and asuitable carrier moistened with an inert liquid diluent.

The compounds of this invention may also be administered rectally in theform of suppositories. These compositions can be prepared by mixing thedrug with a suitable non-irritating excipient which is solid at roomtemperature, but liquid at rectal temperature, and, therefore, will meltin the rectum to release the drug. Such materials include cocoa butter,beeswax, and polyethylene glycols.

Compositions and methods of the invention also may utilize controlledrelease technology. Thus, for example, the disclosed compounds may beincorporated into a hydrophobic polymer matrix for controlled releaseover a period of days. The composition of the invention may then bemolded into a solid implant, or externally applied patch, suitable forproviding efficacious concentrations of the PARP inhibitors over aprolonged period of time without the need for frequent re-dosing. Suchcontrolled release films are well known to the art. Particularlypreferred are transdermal delivery systems. Other examples of polymerscommonly employed for this purpose that may be used in the presentinvention include nondegradable ethylene-vinyl acetate copolymer adegradable lactic acid-glycolic acid copolymers which may be usedexternally or internally. Certain hydrogels such aspoly(hydroxyethylmethacrylate) or poly(vinylalcohol) also may be useful,but for shorter release cycles than the other polymer release systems,such as those mentioned above.

In an embodiment, the carrier is a solid biodegradable polymer ormixture of biodegradable polymers with appropriate time releasecharacteristics and release kinetics. The composition of the inventionmay then be molded into a solid implant suitable for providingefficacious concentrations of the compounds of the invention over aprolonged period of time without the need for frequent re-dosing. Thecomposition of the present invention can be incorporated into thebiodegradable polymer or polymer mixture in any suitable manner known toone of ordinary skill in the art and may form a homogeneous matrix withthe biodegradable polymer, or may be encapsulated in some way within thepolymer, or may be molded into a solid implant.

In one embodiment, the biodegradable polymer or polymer mixture is usedto form a soft “depot” containing the pharmaceutical composition of thepresent invention that can be administered as a flowable liquid, forexample, by injection, but which remains sufficiently viscous tomaintain the pharmaceutical composition within the localized area aroundthe injection site. The degradation time of the depot so formed can bevaried from several days to a few years, depending upon the polymerselected and its molecular weight. By using a polymer composition ininjectable form even the need to make an incision may be eliminated. Inany event, a flexible or flowable delivery “depot” will adjust to theshape of the space it occupies with the body with a minimum of trauma tosurrounding tissues. The pharmaceutical composition of the presentinvention is used in amounts that are therapeutically effective, and maydepend upon the desired release profile, the concentration of thepharmaceutical composition required for the sensitizing effect, and thelength of time that the pharmaceutical composition has to be releasedfor treatment.

The compounds of the invention are used in the composition in amountsthat are therapeutically effective. The compositions may be sterilizedand/or contain adjuvants, such as preserving, stabilizing, welling, oremulsifying agents, solution promoters, salts for regulating the osmoticpressure, and/or buffers. In addition, they may also contain othertherapeutically valuable substances, such as, without limitation, thespecific chemotherapeutic agents recited herein. The compositions areprepared according to conventional mixing, granulating, or coatingmethods, and contain about 0.1 to 75% by weight, preferably about 1 to50% by weight, of the compound of the invention.

To be effective therapeutically as central nervous system targets, thecompounds of the present invention should readily penetrate theblood-brain barrier when peripherally administered. Compounds whichcannot penetrate the blood-brain barrier can be effectively administeredby an intraventricular route or other appropriate delivery systemsuitable for administration to the brain.

For medical use, the amount required of the active ingredient to achievea therapeutic effect will vary with the particular compound, the routeof administration, the mammal under treatment, and the particulardisorder or disease being treated. A suitable systematic dose of acompound of the present invention or a pharmacologically acceptable saltthereof for a mammal suffering from or likely to suffer from, any ofcondition as described hereinbefore is in the range of about 0.1 mg/kgto about 100 mg/kg of the active ingredient compound, the typical dosagebeing about 1 to about 10 mg/kg.

It is understood, however, that a specific dose level for any particularpatient will depend upon a variety of factors including the activity ofthe specific compound employed, the age, body weight, general health,sex, diet, time of administration, rate of excretion, drug combination,and the severity of the particular disease being treated and form ofadministration.

It is understood that the ordinarily skilled physician or veterinarianwill readily determine and prescribe the effective amount of thecompound for prophylactic or therapeutic treatment of the condition forwhich treatment is administered. In so proceeding, the physician orveterinarian can, for example, employ an intravenous bolus followed byan intravenous infusion and repeated administrations, parenterally ororally, as considered appropriate. While it is possible for an activeingredient to be administered alone, it is preferable to present it as aformulation.

When preparing dosage form incorporating the compositions of theinvention, the compounds may also be blended with conventionalexcipients such as binders, including gelatin, pregelatinized starch,and the like; lubricants, such as hydrogenated vegetable oil, stearicacid, and the like; diluents, such as lactose, mannose, and sucrose;disintegrants, such as carboxymethylcellulose and sodium starchglycolate; suspending agents, such as povidone, polyvinyl alcohol, andthe like; absorbants, such as silicon dioxide; preservatives, such asmethylparaben, propylparaben, and sodium benzoate; surfactants, such assodium lauryl sulfate, polysorbate 80, and the like; colorants;flavorants; and sweeteners. Pharmaceutically acceptable excipients arewell known in the pharmaceutical arts and are described, for example, inRemington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa.(e.g., 20^(th) Ed., 2000), and Handbook of Pharmaceutical Excipients,American Pharmaceutical Association, Washington, D.C., (e.g., 1^(st),2^(nd) and 3^(rd) Eds., 1986, 1994, and 2000, respectively).

The present invention relates to the use of a compound of Formula (I) inthe preparation of a medicament for the treatment of any disease ordisorder in an animal described herein. In an embodiment, the compoundsof the present invention are used to treat cancer. In a preferredembodiment, the compounds of the present invention are used topotentiate the cytotoxic effects of ionizing radiation. In such anembodiment, the compounds of the present invention act as aradiosensitizer. In an alternative preferred embodiment, the compoundsof the present invention are used to potentiate the cytotoxic effects ofchemotherapeutic agents. In such an embodiment, the compounds of thepresent invention act as a chemosensitizer. In another preferredembodiment, the compounds of the present invention are used to inhibitthe growth of cells having defects in the homologous recombination (HR)pathway of double-stranded DNA repair.

Any pharmacologically-acceptable chemotherapeutic agent that acts bydamaging DNA is suitable as the chemotherapeutic agent of the presentinvention. In particular, the present invention contemplates the use ofa chemotherapeutically effective amount of at least one chemotherapeuticagent including, but not limited to: temozolomide, adriamycin,camptothecin, carboplatin, cisplatin, daunorubicin, docetaxel,doxorubicin, interferon-alpha, interferon-beta, interferon-gamma,interleukin 2, irinotecan, paclitaxel, topotecan, a taxoid,dactinomycin, danorubicin, 4′-deoxydoxorubicin, bleomycin, pilcamycin,mitomycin, neomycin, gentamycin, etoposide 4-OH cyclophosphamide, aplatinum coordination complex, topotecan, and mixtures thereof.According to a preferred aspect, the chemotherapeutic agent istemozolomide.

The invention contained herein demonstrates the usefulness of thecompounds and compositions of the present invention in treating and/orpreventing cancer, such as by radiosensitizing and/or chemosensitizingtumor and/or cancer cells to chemotherapeutic agents, and to inhibit thegrowth of cells having defects in the homologous recombination (HR)pathway of double-stranded DNA repair.

The following examples are for illustrative purposes only and are notintended to limit the scope of the application.

In one embodiment, the present invention provides a tetraazaphenalen-3-one compound of Formula (I), or a pharmaceutically acceptablesalt thereof:

wherein R is(a) NR¹R², wherein R¹ is selected from the group consisting of hydrogen,C₁-C₆ straight or branched chain alkyl, C₂-C₆ straight or branched chainalkenyl, C₃-C₈ cycloalkyl, C₁-C₆ alkoxy, C₂-C₆ alkenyloxy, phenyl,phenoxy, benzyloxy,NR^(A)R^(B) (C₁-C₆ straight or branched chain alkyl), NR^(A)R^(B) (C₂-C₆straight or branched chain alkenyl), (C₁-C₆ straight or branched chainalkyl)carbonyl, (C₂-C₆ straight or branched chain alkenyl)carbonyl,(C₃-C₈ cycloalkyl)carbonyl,(C₁-C₆ straight or branched chain alkyl)oxycarbonyl, (C₂-C₆ straight orbranched chain alkenyl)oxycarbonyl, (C₃-C₈ cycloalkyl)oxycarbonyl,arylcarbonyl, sulfonyl, arylsulfonyl, aryl(C₁-C₆ straight or branchedchain alkyl), aryl(C₂-C₆ straight or branched chain alkenyl), aryl(C₃-C₈cycloalkyl),(C₁-C₆ straight or branched chain alkyl)aryl, (C₂-C₆ straight orbranched chain alkenyl)aryl, (C₃-C₈ cycloalkyl)aryl, aryl,heterocyclyl, heterocyclyl(C₁-C₆ straight or branched chain alkyl), andheterocyclyl(C₂-C₆ straight or branched chain alkenyl); wherein eachheterocyclyl has between 1 and 7 heteroatoms independently selected fromO, N, or S, and wherein each of R^(A) and R^(B) are independentlyselected from the group consisting of hydrogen, C₁-C₆ straight orbranched chain alkyl, C₂-C₆ straight or branched chain alkenyl, andC₃-C₈ cycloalkyl;and R² is selected from the group consisting of C₁-C₆ straight orbranched chain alkyl, C₂-C₆ straight or branched chain alkenyl, C₃-C₈cycloalkyl, C₁-C₆ alkoxy, C₂-C₆ alkenyloxy, phenyl, phenoxy, benzyloxy,NR^(X)R^(Y) (C₁-C₆ straight or branched chain alkyl), NR^(X)R^(Y) (C₂-C₆straight or branched chain alkenyl), (C₁-C₆ straight or branched chainalkyl)carbonyl, (C₂-C₆ straight or branched chain alkenyl)carbonyl,(C₃-C₈ cycloalkyl)carbonyl, (C₁-C₆ straight or branched chainalkyl)oxycarbonyl, (C₂-C₆ straight or branched chainalkenyl)oxycarbonyl,(C₃-C₈ cycloalkyl)oxycarbonyl, arylcarbonyl, sulfonyl, arylsulfonyl,aryl(C₁-C₆ straight or branched chain alkyl), aryl(C₂-C₆ straight orbranched chain alkenyl), aryl(C₃-C₈ cycloalkyl), (C₁-C₆ straight orbranched chain alkyl)aryl, (C₂-C₆ straight or branched chainalkenyl)aryl, (C₃-C₈ cycloalkyl)aryl, aryl,heterocyclyl, heterocyclyl(C₁-C₆ straight or branched chain alkyl), andheterocyclyl(C₂-C₆ straight or branched chain alkenyl); wherein eachheterocyclyl has between 1 and 7 heteroatoms independently selected fromO, N, or S, and wherein each of R^(X) and R^(Y) are independentlyselected from the group consisting of hydrogen, C₁-C₆ straight orbranched chain alkyl, C₂-C₆ straight or branched chain alkenyl, andC₃-C₈ cycloalkyl;wherein R¹ and R² are independently substituted with between 0 and 4substituents, each independently selected from halo, C₁-C₆ straight orbranched chain alkyl, C₂-C₆ straight or branched chain alkenyl, C₁-C₆alkoxy, trifluoromethyl, trifluoroethyl, and amino; and provided that R¹and R² may not both be methyl, and R² may not be (phenyl)prop-1-yl whenR¹ is hydrogen; or(b) aryloxy, substituted with between 0 and 4 substituents, eachindependently selected from the group consisting of halo, C₁-C₆ alkoxy,trifluoromethyl, trifluoroethyl, C₁-C₆ straight or branched chain alkyl,C₂-C₆ straight or branched chain alkenyl, C₃-C₈ cycloalkyl, NR^(C)R^(D),NR^(C)R^(D)(C₁-C₆ straight or branched chain alkyl), andNR^(C)R^(D)(C₂-C₆ straight or branched chain alkenyl), wherein each ofR^(C) and R^(D) is independently selected from the group consisting ofhydrogen, C₁-C₆ straight or branched chain alkyl, C₂-C₆ straight orbranched chain alkenyl, and C₃-C₈ cycloalkyl; and when more than onesubstituent is of the form NR^(C)R^(D), each occurrence of R^(C) andR^(D) is independently selected from the group consisting of hydrogen,C₁-C₆ straight or branched chain alkyl, C₂-C₆ straight or branched chainalkenyl, and C₃-C₈ cycloalkyl; or(c) a heterocyclyl having between 1 and 7 heteroatoms independentlyselected from O, N, or S; and having between 0 and 4 substituentsindependently selected from the group consisting of halo, haloalkyl,hydroxyl, nitro, trifluoromethyl, trifluoroethyl, C₁-C₆ straight orbranched chain alkyl, C₂-C₆ straight or branched chain alkenyl, C₁-C₆alkoxy, C₂-C₆ alkenyloxy, phenyl, phenoxy, benzyloxy, amino,thiocarbonyl, cyano, imino, NR^(E)R^(F)(C₁-C₆ straight or branched chainalkyl), NR^(E)R^(F)(C₂-C₆ straight or branched chain alkenyl)sulflhydryl, thioalkyl, dioxa-spiroethyl, (C₁-C₆ straight or branchedchain alkyl) carbonyl, (C₂-C₆ straight or branched chainalkenyl)carbonyl, (C₁-C₆ straight or branched chain alkyl)oxycarbonyl,(C₂-C₆ straight or branched chain alkenyl)oxycarbonyl, arylcarbonyl,sulfonyl, arylsulfonyl, aryl(C₁-C₆ straight or branched chain alkyl),aryl(C₂-C₆ straight or branched chain alkenyl), aryl(C₃-C₈ cycloalkyl),(C₁-C₆ straight or branched chain alkyl)aryl, (C₂-C₆ straight orbranched chain alkenyl)aryl, (C₃-C₈ cycloalkyl)aryl, aryl, heterocyclyl,heterocyclyl(C₁-C₆ straight or branched chain alkyl), andheterocyclyl(C₂-C₆ straight or branched chain alkenyl), wherein eachheterocyclyl has between 1 and 7 heteroatoms independently selected fromO, N, or S, wherein each of R^(E) and R^(F) is independently selectedfrom the group consisting of hydrogen, C₁-C₆ straight or branched chainalkyl, C₂-C₆ straight or branched chain alkenyl, and C₃-C₈ cycloalkyl;and when more than one substituent is of the form NR^(E)R^(F) eachoccurrence of R^(E) and R^(F) is independently selected from the groupconsisting of hydrogen, C₁-C₆ straight or branched chain alkyl, C₂-C₆straight or branched chain alkenyl, and C₃-C₈ cycloalkyl; wherein eachof said 0-4 substituents is independently substituted with between 0 and4 further substituents, and each said further substituent isindependently selected from halo, C₁-C₆ straight or branched chainalkyl, C₂-C₆ straight or branched chain alkenyl, C₃-C₈ cycloalkyl, C₁-C₆alkoxy, trifluoromethyl, trifluoroethyl, and amino; provided that R hasat least one substituent when R is an N-piperidinyl, N-pyrrolidinyl oran N-morpholinyl group.

In some embodiments each ring of each heterocycle of Formula (I) isindependently 5-7 atoms in size.

Some embodiments include one, two or three nitrogen atoms in at leastone ring of the heterocycle of Formula (I).

In some embodiments, the heterocyclyl of Formula (I) comprises 1-3rings. In some embodiments, the heterocyclyl has 1-7 heteroatomsindependently selected from O, N, and S.

In some embodiments, the heterocyclyl of Formula (I) is selected fromthe group consisting of piperidinyl, piperazinyl, pyridazinyl,dihydropyridyl, tetrahydropyridyl, pyridinyl, pyrimidinyl,dihydropyrimidinyl, tetrahydrophyrimidinyl, hexahydropyrimidinyl,dihydropyrazinyl, tetrahydropyrazinyl, pyrrolidinyl, imidazolidinyl,pyrazolidinyl, pyrrolyl, dihydropyrolyl, imidazolyl, dihydroimidazoyl,pyrazolyl, dihydropyrazolyl, azepanyl, [1,2]diazepanyl, [1,3]diazepanyl,[1,4]diazepanyl, indolyl, dihydroindolyl, isoindolyl, dihydroisoindoly,dihydroquinolyl, tetrahydroquinolyl, dihydroisoquinolyl, andtetrahydroisoquinolyl.

In another embodiment, the present invention provides a compoundselected from the group consisting of

and pharmaceutically acceptable salts thereof.

In some embodiments the invention provides the compound which is

or a pharmaceutically acceptable salt thereof.

In some embodiments the invention provides the compound which is

or a pharmaceutically acceptable salt thereof.

In some embodiments the present invention provides a method ofchemosensitizing cancer cells in a mammal in need of chemotherapy,comprising administering to said mammal a compound of Formula (I) asdescribed herein, or a pharmaceutically acceptable salt thereof. In someembodiments, said mammal is a human. In some embodiments, saidadministration is administration of a pharmaceutical compositioncomprising said compound and a pharmaceutically acceptable carrier. Insome embodiments, the chemosensitization method further comprisesadministering to said mammal a chemotherapeutic agent. In someembodiments, said chemosensitizing compound and said chemotherapeuticagent are administered essentially simultaneously.

In some embodiments the present invention provides a method ofchemosensitizing cancer cells in a mammal in need of chemotherapy,comprising administering to said mammal a compound selected from thegroup consisting of compounds 7-28, 30-46, 50-66, 69, 72, 74-76, andpharmaceutically acceptable salts thereof, as described herein. In someembodiments, said mammal is a human. In some embodiments, saidadministration is administration of a pharmaceutical compositioncomprising said compound and a pharmaceutically acceptable carrier. Insome embodiments, the chemosensitization method further comprisesadministering to said mammal a chemotherapeutic agent. In someembodiments, said chemosensitizing compound and said chemotherapeuticagent are administered essentially simultaneously.

In some embodiments, the chemotherapeutic agent of the invention isselected is selected from the group consisting of temozolomide,adriamycin, camptothecin, carboplatin, cisplatin, daunorubicin,docetaxel, doxorubicin, interferon-alpha, interferon-beta,interferon-gamma, interleukin 2, irinotecan, paclitaxel, topotecan, ataxoid, dactinomycin, danorubicin, 4′-deoxydoxorubicindeoxydoxorubicin,bleomycin, pilcamycin, mitomycin, neomycin, gentamycin, etoposide, 4-OHcyclophosphamide, a platinum coordination complex, and mixtures thereof.In some embodiments, the chemotherapeutic agent is temozolomide or asalt thereof.

In some embodiments, the present invention provides a method ofradiosensitizing cancer cells in a mammal in need of radiation therapycomprising administering to said mammal a compound of Formula (I) asdescribed herein, or a pharmaceutically acceptable salt thereof. In someembodiments, said mammal is a human. In some embodiments, saidadministration is administration of a pharmaceutical compositioncomprising said compound and a pharmaceutically acceptable carrier.

In some embodiments, the present invention provides a method ofradiosensitizing cancer cells in a mammal in need of radiation therapycomprising administering to said mammal a compound selected from thegroup consisting of compounds 7-28, 30-46, 50-66, 69, 72, 74-76, andpharmaceutically acceptable salts thereof, as described herein. In someembodiments, said mammal is a human. In some embodiments, saidadministration is administration of a pharmaceutical compositioncomprising said compound and a pharmaceutically acceptable carrier.

In some embodiments, the invention provides a pharmaceutical compositioncomprising a compound of Formula (I) as described herein, or apharmaceutically acceptable salt thereof, and a pharmaceuticallyacceptable carrier. In some embodiments, the pharmaceutical compositionfurther comprises a chemotherapeutic agent as described herein.

In some embodiments, the invention provides a pharmaceutical compositioncomprising a compound selected from the group consisting of compounds7-28, 30-46, 50-66, 69, 72, 74-76, and pharmaceutically acceptable saltsthereof, as described herein. In some embodiments, the pharmaceuticalcomposition further comprises a chemotherapeutic agent as describedherein.

In some embodiments, the cancer cells treated by the chemosensitizingand/or radiosensitizing methods of the invention are selected from thegroup consisting of ACTH-producing tumors, acute lymphocytic leukemia,acute nonlymphocytic leukemia, cancer of the adrenal cortex, bladdercancer, brain cancer, breast cancer, cervical cancer, chroniclymphocytic leukemia, chronic myelocytic leukemia, colorectal cancer,cutaneous T-cell lymphoma, endometrial cancer, esophageal cancer,Ewing's sarcoma, gallbladder cancer, hairy cell leukemia, head and neckcancer, Hodgkin's lymphoma, Kaposi's sarcoma, kidney cancer, livercancer, lung cancer (small and/or non-small cell), malignant peritonealeffusion, malignant pleural effusion, melanoma, mesothelioma, multiplemyeloma, neuroblastoma, non-Hodgkin's lymphoma, osteosarcoma, ovariancancer, ovary (germ cell) cancer, prostate cancer, pancreatic cancer,penile cancer, retinoblastoma, skin cancer, soft-tissue sarcoma,squamous cell carcinomas, stomach cancer, testicular cancer, thyroidcancer, trophoblastic neoplasms, uterine cancer, vaginal cancer, cancerof the vulva and Wilm's tumor. In some embodiments, the cancer cellstreated by the chemosensitizing and/or radiosensitizing methods of theinvention are selected from the group consisting of brain cancer,melanoma, head and neck cancer, testicular cancer, ovarian cancer,breast cancer, non small cell lung cancer, and rectal cancer.

In some embodiments, the invention provides a method of treating amammal having a cancer characterized by having a defect in thehomologous recombination (HR) pathway of double-stranded DNA repair,comprising administering to said mammal a compound of Formula (I) asdescribed herein, or a pharmaceutically acceptable salt thereof. In someembodiments, said mammal is a human. In some embodiments, saidadministration is administration of a pharmaceutical compositioncomprising said compound and a pharmaceutically acceptable carrier. Insome embodiments, the cancer cells have a phenotype selected from thegroup consisting of i) a BRCA-1 defect, ii) a BRCA-2 defect, iii) aBRCA-1 and BRCA-2 defect, and iv) Fanconi anemia. In some embodiments,the cancer cells are selected from breast cancer or ovarian cancer.

In some embodiments, the invention provides a method of treating amammal having a cancer characterized by having a defect in thehomologous recombination (HR) pathway of double-stranded DNA repair,comprising administering to said mammal a compound selected from thegroup consisting of compounds 7-28, 30-46, 50-66, 69, 72, 74-76, andpharmaceutically acceptable salts thereof, as described herein. In someembodiments, said mammal is a human. In some embodiments, saidadministration is administration of a pharmaceutical compositioncomprising said compound and a pharmaceutically acceptable carrier. Insome embodiments, the cancer cells have a phenotype selected from thegroup consisting of i) a BRCA-1 defect, ii) a BRCA-2 defect, iii) aBRCA-1 and BRCA-2 defect, and iv) Fanconi anemia. In some embodiments,the cancer cells are selected from breast cancer or ovarian cancer.

Synthetic Procedures for the Disclosed Compounds

Procedure A: Preparation of 3-nitro-phthalic Acid Dimethyl Ester, 2

To a stirred solution of 4-nitro-isobenzofuran-1,3-dione (150 g, 0.78mol), 1, in 2 L of MeOH was added 50 mL of concentrated sulfuric acid.The reaction was heated to reflux for 16 hours. The mixture solution wascooled to room temperature and then poured into 3 L of ice water andresulted in a heavy white precipitate. This was triturated for 15minutes and the precipitated was filtered off and the solid was washedwith water thoroughly and dried to afford 120 g of 3-nitro-phthalic aciddimethyl ester, 2, as a white solid (65%). ¹H NMR (300 MHz, DMSO-d₆):8.54 (d, J=7.25 Hz, 1H), 8.42 (d, J=7.82 Hz, 1H), 7.98 (t, J=8.20 Hz,1H), 3.99 (s, 3H), 3.98 (s, 3H). ¹³C NMR: 52.03, 52.29, 111.02, 115.67,119.08, 131.80, 133.68, 148.80, 167.64, 168.63.

Procedure B: Preparation of 3-amino-phthalic Acid Dimethyl Ester, 3

The compound 2 (205 g, 1.0 mol) was dissolved in 2 L of MeOH. Catalytic10% Pd/C was added and the solution was hydrogenated under H₂ (45 psi)on a Parr hydrogenation apparatus at room temperature overnight.Filtered through celite and evaporated to give a quantitative yield of3-amino-phthalic acid dimethyl ester, 3. ¹H NMR (300 MHz, DMSO-d₆): 7.26(t, J=7.33 Hz, 1H), 6.94 (d, J=8.34 Hz, 1H), 6.77 (d, J=8.33 Hz, 1H),6.12 (s, 2H), 3.77 (s, 3H), 3.76 (s, 3H). ¹³C NMR: 51.51, 51.77, 110.50,115.16, 118.56, 131.26, 133.16, 148.28, 167.12, 168.11.

Procedure C: Preparation of2-chloromethyl-4-oxo-3,4-dihydro-quinazoline-5-carboxylic Acid MethylEster, 4

100 mL of chloroacetonitrile was set stirring in 130 mL of 1,4 dioxaneat room temperature. Dry HCl gas was bubbled through the solution forthirty minutes followed by the addition of 30 g of 3-amino-1,2-phthalicacid dimethyl ester, 3. The reaction was refluxed for approximatelythree hours, resulting in a heavy white precipitate. The suspension wascooled with an ice bath, filtered and washed with pentane to remove anyresidual solvents. 30 g (83%) of an analytically pure white solid, 4,was isolated. ¹H NMR (300 MHz, DMSO-d₆): 7.88 (t, J=8.33 Hz, 1H), 7.79(d, J=7.08 Hz, 1H), 7.52 (d, J=7.33 Hz, 1H), 4.60 (s, 2H), 3.84 (s, 3H);¹³C NMR: 42.21, 54.86, 119.95, 127.77, 130.86, 135.71, 136.78, 150.59,155.70, 162.49, 171.24.

General Procedure D: Preparation of Compounds, 5

Displacement of the chloro group of compound 4 with nucleophiles such asamine using General procedure D provides the compounds 5. To a solutionof the chloro compound 4 in dry DMF or MeCN is added potassium carbonateand a nucleophile such as an amine. The reaction mixture is heated to70° C. for 12 hours and cooled to room temperature. Water is added tothe reaction mixture, followed by ethyl acetate. The organic layer iscollected, washed with water, brine and dried over sodium sulfate. Thesolvents are removed in vacuum. The residue is purified by columnchromatography on silica gel using ethyl acetate/hexanes as eluent togive the products 5 in 50-95% yield. An example was given in thepreparation of compound 7.

General Procedure E: Preparation of Compounds, 6

A 2,9-Dihydro-1,2,7,9-tetraaza-phenalen-3-one ring can be formed bycondensation of the compounds 6 with hydrazine. To a solution of thecompounds 6 in absolute ethanol is added excess anhydrous hydrazine atroom temperature. The solution is refluxed for overnight and cooled toroom temperature. Ice-cold water is added and white solid is separated.The solid is collected by vacuum filtration and washed with water andsmall amount of methanol to give white solid products 6 in 40-90% yield.An example was given in the preparation of compound 7.

Example 1 Preparation of8-(4-hydroxy-piperidin-1-ylmethyl)-2,9-dihydro-1,2,7,9-tetraaza-phenalen-3-one,7

Following the General Procedure D: A solution of MeCN (25 ml),4-hydroxypiperidine (0.46 mg, 4.5 mmol), 4 (1.0 g, 3.9 mmol), andpotassium carbonate (1 g, 7 mmol) was set refluxing under nitrogen andstirred overnight. Reaction mixture was evaporated to dryness andextracted with dichloromethane. Purified with a silica column using 9:1dichloromethane/MeOH to afford 1.05 g (84%) of an off-white solid,2-(4-Hydroxy-piperidin-1-ylmethyl)-4-oxo-3,4-dihydro-quinazoline-5-carboxylicacid methyl ester, 7a.

Following the General Procedure E: To a solution of compound 7a (1.0 g,3.1 mmol) in EtOH (20 mL) when refluxing was added hydrazine monohydrate(7 mL, large excess) and heated overnight. Reaction was cooled to RT andH₂O (15 mL) was added resulting in a heavy white precipitate. Filteredand washed with 1:1 EtOH/H₂O to afford 0.6 g (64%) of an analyticallypure white solid, 7. MP: 168-171° C.; MS (ES+): 300; ¹H NMR (300 MHz,CD₃OD): 1.46-1.55 (m, 2H), 1.71-1.75 (m 2H) 2.15-2.23 (m 2H) 2.70-2.75(m, 2H) 3.16-3.18 (m, 1H) 3.25 (s, 2H) 3.47-3.55 (m, 1H) 7.30-7.33 (m,1H) 7.60-7.64 (m, 2H). Anal. Calcd. for C₁₅H₁₇N₅O₂.1.7 H₂O: C, 56.45; H,6.06; N, 21.94; Found: C, 56.10; H, 6.00; N, 22.25.

The compound 7 can be formulated with an acid. For example: to asolution of 7 (0.6 g, 2.0 mmol) in 10 mL of 1,4 dioxane/DMF (9:1) at 90°C. was added MsOH (0.14 mL, 2.1 mmol) resulting in a heavy whiteprecipitate. Filtered and triturated in diethyl ether to afford 0.5 g(63%) of an off-white solid, mesylate salt of 7. ¹H NMR (300 MHz,DMSO-d₆): 1.55-1.58 (m 2H), 1.78-1.82 (m, 2H), 2.15 (s, 3H), 3.15-3.50(m 4H), 3.63-3.65 (m, 1H), 4.04 (s, 2H), 7.24 (d, J=8.5 Hz, 1H),7.51-7.66 (m 2H), 11.73 (s, 1H)

Anal. Calcd. for C₁₅H₁₇N₅O₂.1CH₃SO₃H. 2H₂O: C, 44.54; H, 5.84; N, 16.23,S, 7.43; Found: C, 44.48; H, 5.76; N, 16.27, S, 7.60.

The following compounds were synthesized from the similar procedures ofpreparation of compound 7, using the appropriate corresponding amines.

Preparation of8-(4-phenyl-piperazin-1-ylmethyl)-2,9-dihydro-1,2,7,9-tetraaza-phenalen-3-one,8

Synthesized using 1-phenylpiperazine for General Procedure D. 52%overall yield for last two steps. MS (ES+): 361; ¹H NMR (300 MHz,DMSO-d₆): 2.65-2.68 (m, 4H), 3.19-3.22 (m, 4H) 3.39 (s, 2H); 6.78 (t,J=7.2 Hz, 1H); 6.95 (d, J=8.0 Hz, 2H), 7.19 (t, J=7.2 Hz, 2H), 7.48-7.51(m, 1H), 7.62-7.64 (d, J=7.2 Hz, 1H), 7.75 (t, J=8.0 Hz, 1H), 11.23 (s,br, 1H), 11.78 (s, 1H); Anal. Calcd. for C₂₀H₂₀N₆O₁.2.0H₂O: C, 60.59; H,6.10; N, 21.20; Found: C, 60.48; H, 6.05; N, 21.35.

Preparation of8-(4-benzyl-piperidin-1-ylmethyl)-2,9-dihydro-1,2,7,9-tetraaza-phenalen-3-one,9

Synthesized using 1-benzylpiperazine for General Procedure D. 20%overall yield for last two steps. MS (ES−): 372; ¹H NMR (300 MHz,DMSO-d₆): 1.22-1.50 (m, 5H), 2.45-2.55 (m, 4H), 2.85 (d, 2H), 3.28 (s,2H), 7.14-7.19 (m, 3H), 7.25-7.30 (m 2H), 7.50 (d, J=7.0 Hz, 1H), 7.62(d, J=7.7 Hz, 1H), 7.75 (t, J=7.7 Hz, 1H), 11.25 (s, br, 1H), 11.76 (s,1H); Anal. Calcd. for C₂₂H₂₃N₅O₁: C, 70.76; H, 6.21; N, 18.75; Found: C,70.36; H, 6.18; N, 18.63.

Preparation of8-phenoxymethyl-2,9-dihydro-1,2,7,9-tetraaza-phenalen-3-one, 10

Synthesized using phenol for General Procedure D. 60% overall yield forlast two steps. MS (ES+): 293; ¹H NMR (300 MHz, DMSO-d₆): 4.90 (s, br,3H), 7.00 (t, J=6.6 Hz, 1H), 7.08 (d, J=8.2 Hz, 2H), 7.34 (t, J=7.7 Hz,2H), 7.45 (d, J=7.7 Hz, 1H), 7.65 (d, J=7.7 Hz, 1H), 7.76 (t, J=7.2 Hz,1H), 11.20 (s, br, 1H), 11.80 (s, 1H). Anal. Calcd. for C₁₆H₁₂N₄O₂.0.75H₂O. 0.25 N₂H₄: C, 61.24; H, 4.66; N, 20.08; Found: C, 61.06; H, 4.27;N, 20.13;

Preparation of8-(4-(4-fluoro-phenyl)-3,6-dihydro-2H-pyridin-1-ylmethyl)-2,9-dihydro-1,2,7,9-tetraaza-phenalen-3-one,11

Synthesized using 4-(4-fluorophenyl)-1,2,3,6-tetrahydropyridinehydrochloride for General Procedure D. 24% overall yield for last twosteps. MS (ES+): 376; ¹H NMR (400 MHz, DMSO-d₆): 2.51-2.53 (s, br, 2H),2.77 (t, J=5.4 Hz, 2H), 3.24 (s, br, 2H), 3.46 (s, 2H), 6.16 (m, 1H),7.16 (t, J=8.8 Hz, 2H), 7.46-7.52 (m, 3H), 7.63 (d, J=7.8 Hz, 1H), 7.44(t, J=7.8 Hz, 1H), 11.18 (s, br, 1H), 11.79 (s, 1H). A mesylate salt of11 was prepared. ¹H NMR (400 MHz, DMSO-d₆): 2.34 (s, 3H), 2.84 (bs, 2H),3.66 (m 2H), 4.11 (m, 2H), 4.36 (s, 2H), 6.21 (m, 1H), 7.25 (t, J=8.8Hz, 2H), 7.43 (d, J=7.4 Hz, 1H), 7.56-7.59 (m, 2H), 7.72 (d, J=7.4 Hz,1H), 7.82 (t, J=7.5 Hz, 1H), 11.25 (s, br, 1H), 11.76 (s, 1H). Anal.Calcd. for C₂₁H₁₈FN₅O₁.1.0 CH₃SOH. 0.2 H₂O: C, 55.62; H, 4.75; N, 14.74;S, 6.75; Found: C, 55.65; H, 4.71; N, 14.73; S, 6.74.

Preparation of8-[4-(4-chloro-phenyl)-piperazin-1-ylmethyl]-2,9-dihydro-1,2,7,9-tetraaza-phenalen-3-one,12

Synthesized using 1-(4-chlorophenyl)-piperazine for General Procedure D.23% overall yield for last two steps. A mesylate salt of 12 wasprepared. MS (ES+): 396; ¹H NMR (400 MHz, DMSO-d₆): 2.33 (s, 3H), 4.31(s, 2H), 7.03 (d, J=9.3 Hz, 2H), 7.31 (d, J=9.3 Hz, 2H), 7.43 (d, J=8.5Hz, 1H), 7.72 (d, J=8.5 Hz, 1H), 7.82 (t, J=7.9 Hz, 1H), 11.23 (s, br,1H), 11.90 (s, 1H). Anal. Calcd. for C₂₀H₁₉ClN₆O₁.1.0 CH₃SOH: C, 51.37;H, 4.72; N, 17.12; S, 6.53; Found: C, 51.27; H, 4.91; N, 17.03; S, 6.48.

Preparation of8-(4-phenyl-3,6-dihydro-2H-pyridin-1-ylmethyl)-2,9-dihydro-1,2,7,9-tetraaza-phenalen-3-one,13

Synthesized using 4-phenyl-1,2,3,6-tetrahydro-pyridine for GeneralProcedure D. 80% overall yield for last two steps. MS (ES+): 358; ¹H NMR(400 MHz, DMSO-d₆): 2.56 (m, 2H), 2.78 (t, J=5.5 Hz, 2H), 3.25 (d, J=2.6Hz, 2H), 3.47 (s, 2H), 6.19 (s, 1H), 7.23-7.27 (m, 1H), 7.24 (t, J=7.6Hz, 2H), 7.45 (d, J=7.1 Hz, 2H), 7.51 (d, J=8.9 Hz, 1H), 7.62 (d, J=7.1Hz, 1H), 7.75 (t, J=8.0 Hz, 1H), 11.27 (s, br, 1H), 11.78 (s, 1H). Amesylate salt of 13 was prepared. ¹H NMR (400 MHz, DMSO-d₆): 2.34 (s,3H), 2.84-2.88 (m, 2H), 3.65-3.69 (m, 2H), 4.13 (s, 2H), 4.37 (s, 2H),6.21-6.25 (m, 1H), 7.32-7.44 (m, 4H), 7.53 (d, J=8.6 Hz, 2H), 7.72 (d,J=7.3 Hz, 1H), 7.82 (t, J=8.1 Hz, 1H), 11.30 (s, br, 1H), 11.93 (s, 1H).Anal. Calcd. for C₂₁H₁₉N₅O. 1.0 CH₃SOH. 0.4 H₂O: C, 57.35; H, 5.21; N,15.20; S, 6.96; Found: C, 57.30; H, 5.16; N, 15.29; S, 7.10;

Preparation of8-[(3,4-dichloro-benzylamino)-methyl]-2,9-dihydro-1,2,7,9-tetraaza-phenalen-3-one,14

Synthesized using 3,4-dichlorobenzylamine for General Procedure D. 10%overall yield for last two steps. A mesylate salt of 14 was prepared. MS(ES+): 375; ¹H NMR (300 MHz, DMSO-d₆): 2.33 (s, 3H), 4.06 (s, 2H), 4.33(s, 2H), 7.39 (d, J=8.0 Hz, 1H), 7.53-7.57 (m, 1H), 7.69-7.88 (m, 4H),11.31 (s, br, 1H), 11.91 (s, 1H).

Preparation of8-{[2-(3-Fluoro-phenyl)-ethylamino]-methyl}-2,9-dihydro-1,2,7,9-tetraaza-phenalen-3-one,15

Synthesized using 3-fluorophenethylamine for General Procedure D. 12%overall yield for last two steps. A mesylate salt of 15 was prepared. MS(ES+): 338; ¹H NMR (300 MHz, DMSO-d₆): 2.34 (s, 3H), 3.02-3.08 (m, 2H),3.34-3.38 (m, 2H), 4.14 (s, 2H), 7.08-7.18 (m, 3H), 7.37-7.44 (m, 2H),7.71 (d, J=7.8 Hz, 1H), 7.82 (t, J=7.8 Hz, 1H), 11.92 11.35 (s, br, 1H),(s, 1H).

Preparation of8-[(3-trifluoromethyl-benzylamino)-methyl]-2,9-dihydro-1,2,7,9-tetraaza-phenalen-3-one,16

Synthesized using 3-(trifluoromethyl)benzylamine for General ProcedureD. 14% overall yield for last two steps. A mesylate salt of 16 wasprepared. MS (ES+): 374; ¹H NMR (300 MHz, DMSO-d₆): 2.33 (s, 3H), 4.10(s, 2H), 4.43 (s, 2H), 7.39 (d, J=7.6 Hz, 1H), 7.69-7.86 (m, 5H), 7.99(s, 1H), 11.25 (s, br, 1H), 11.91 (s, 1H). Anal. Calcd. for C₁₉H₁₈F₃N₅O.1.0 CH₃SOH. 1.0 H₂O: C, 46.82; H, 4.14; N, 14.37; S, 6.58; Found: C,46.81; H, 4.17; N, 14.64; S, 6.35.

Preparation of8-(1,4-dioxa-8-aza-spiro[4.5]dec-8-ylmethyl)-2,9-dihydro-1,2,7,9-tetraaza-phenalen-3-one,17

Synthesized using 4-piperidone ethylene ketal for General Procedure D.10% overall yield for last two steps. MS (ES−): 370; ¹H NMR (300 MHz,DMSO-d₆): 1.69-1.71 (m, 4H), 2.57 (s, br, 4H), 3.35 (s, 2H), 3.87 (s,4H), 7.51 (d, J=7.8 Hz, 1H), 7.62 (d, J=7.7 Hz, 1H), 7.74 (t, J=7.8 Hz,1H), 11.23 (s, br, 1H), 11.76 (s, 1H). Anal. Calcd. for C₁₇H₁₉N₅O₃.0.2H₂O: C, 59.19; H, 5.67; N, 20.30; Found: C, 59.03; H, 5.60; N, 20.63.

Preparation of8-{[2-(3,4-dichloro-phenyl)-ethylamino]-methyl}-2,9-dihydro-1,2,7,9-tetraaza-phenalen-3-one,18

Synthesized using 3,4-dichlorophenethylamine for General Procedure D.17% overall yield for last two steps. A mesylate salt of 18 wasprepared. MS (ES−): 387; ¹H NMR (300 MHz, DMSO-d₆): 2.36 (s, 3H), 3.04(t, J=8.2 Hz, 2H), 3.37 (t, J=8.1 Hz, 2H), 4.14 (s, 2H), 7.30-7.43 (m,2H), 7.61-7.75 (m, 3H), 7.79-7.84 (m, 1H), 11.31 (s, br, 1H), 11.91 (s,1H).

Preparation of8-{[2-(3-trifluoromethyl-phenyl)-ethylamino]-methyl}-2,9-dihydro-1,2,7,9-tetraaza-phenalen-3-one,19

Synthesized using 2-(3-Trifluoromethyl-phenyl)-ethylamine for GeneralProcedure D. 39% overall yield for last two steps. A mesylate salt of 19was prepared. MS (ES−): 387; ¹H NMR (300 MHz, DMSO-d₆): 3.74 (s, 3H),3.13 (t, J=8.1 Hz, 2H), 3.30 (t, J=8.2 Hz, 2H), 4.15 (s, 2H), 7.40-7.43(m, 1H), 7.62-7.72 (m, 4H), 7.79-7.85 (m, 1H), 11.35 (s, br, 1H), 11.92(s, 1H).

Preparation of8-[(1-Aza-bicyclo[2.2.2]oct-3-ylamino)-methyl]-2,9-dihydro-1,2,7,9-tetraaza-phenalen-3-one,20

Synthesized using (S)-(−)-3-aminoquinuclidine for General Procedure D.23% overall yield for last two steps. A mesylate salt of 20 wasprepared. MS (ES+): 325; ¹H NMR (300 MHz, DMSO-d₆): 1.97-2.03 (m, 3H),2.20-2.35 (m, 1H), 2.35-2.44 (m, 2H), 2.42 (s, 3H), 3.72-3.80 (m, 6H),4.15-4.21 (m, 1H), 4.38 (s, 2H), 7.46 (d, J=7.6, 1H) 7.69-7.72 (m, 1H),7.78-7.84 (m, 1H), 8.63 (s, br, 3H).

Preparation of8-(4-ethyl-piperazin-1-ylmethyl)-2,9-dihydro-1,2,7,9-tetraaza-phenalen-3-one,21

Synthesized using ethylpiperazine for General Procedure D. 35% overallyield for last two steps. A mesylate salt of 21 was prepared. MS (ES+):313; ¹H NMR (300 MHz, DMSO-d₆): 1.25, (t, J=7.4 Hz, 3H), 2.41 (s, 6H),2.51-3.87 (m, 10H), 3.87 (s, 2H), 7.70 (d, J=8.0 Hz, 1H), 7.81 (d, J=7.9Hz, 1H), 7.91 (t, J=8.1 Hz, 1H), 9.82 (s, 1H), 11.96 (s, 1H). ¹³C NMR(DMSO-d₆): 157.40, 155.99, 140.65, 135.96, 133.84, 126.72, 119.71,118.65, 115.85, 56.09, 50.30, 49.05, 48.66, 8.51. Anal. Calcd. forC₁₆H₂₀N₆O. 2.0 CH₃SO₃H. 1.2 H₂O.: C, 40.84; H, 5.43; N, 15.79; Found: C,41.09; H, 5.82; N, 15.97.

Preparation of8-(4-methyl-piperazin-1-ylmethyl)-2,9-dihydro-1,2,7,9-tetraaza-phenalen-3-one,22

Synthesized using methylpiperazine for General Procedure D. 29% overallyield for last two steps. A mesylate salt of 22 was prepared. MS (ES+):299; ¹H NMR (400 MHz, DMSO-d₆): 2.38 (s, 3H), 2.58-2.63 (m, 2H),3.09-3.18 (m, 4H), 3.40-3.45 (m, 2H), 3.51 (s, 2H), 7.50 (d, J=7.8 Hz,1H), 7.67 (d, J=7.8 Hz, 1H), 7.79 (t, J=7.8 Hz, 1H), 9.53 (s, br, 1H),11.85 (s, 1H). Anal. Calcd. for C₁₅H₁₈N₆O. 1.15 CH₃SO₃H. 1.0 H₂O.: C,45.44; H, 5.81; N, 19.69; S, 8.64; Found: C, 45.18; H, 5.88; N, 19.83;S, 8.68;

Preparation of8-(4-benzyl-[1,4]diazepan-1-ylmethyl)-2,9-dihydro-1,2,7,9-tetraaza-phenalen-3-one,23

Synthesized using 1-benzyl-[1,4]diazepane for General Procedure D. 24%overall yield for last two steps. MP: 140-142° C.; MS (ES−): 387; ¹H NMR(400 MHz, CDCl₃): 1.88 (m, 2H), 2.77 (m, 4H), 2.89 (m, 4H), 3.62 (s,2H), 3.69 (s, 2H), 7.20-7.42 (m, 6H), 7.45 (s, br, 1H), 7.74 (t, J=7.8Hz, 1H), 7.87 (d, J=7.6 Hz, 1H), 11.50 (s, br, 1H); Anal. Calcd. forC₂₂H₂₄N₆O. 1.35 H₂O.: C, 64.01; H, 6.52; N, 20.36; Found: C, 64.18; H,6.59; N, 20.46.

An HCl salt of 23 was prepared: to a solution of 23 (0.5 g) in 20 mL ofdioxane was bubbled HCl gas for 30 min. The solution was stirred at roomtemperature overnight. After filtration, the precipitate was washed withdioxane to afford 0.25 g (48%) of analytically pure off white solid, anHCl salt of 23. ¹H NMR (400 MHz, D₂O): 2.08 (m, 2H), 3.36 (m, 4H), 3.56(m, 4H), 4.04 (s, 2H), 4.24 (s, 2H), 7.02 (d, 1H), 7.20-7.35 (m, 5H);7.36 (d, 1H), 7.45 (t, 1H); Anal. Calcd. for C₂₂H₂₄N₆O. 2.0HCl. 1.15H₂O.: C, 54.81; H, 5.92; N, 17.43; Found: C, 54.81; H, 5.92; N, 17.36.

Preparation of4-(3-oxo-2,9-dihydro-3H-1,2,7,9-tetraaza-phenalen-8-ylmethyl)-[1,4]diazepane-1-carboxylicAcid Tert-Butyl Ester, 24

Synthesized using [1,4]diazepane-1-carboxylic acid t-butyl ester forGeneral Procedure D. 30% overall yield for last two steps. MP: 219-221°C.; MS (ES−): 397; ¹H NMR (400 MHz, CDCl₃): 1.46 (s, 9H); 1.88 (m, 2H);2.83 (m, 4H); 3.50 (m, 4H); 3.59 (s, 2H); 7.63 (m, 1H), 7.72-7.86 (m,3H), 11.90 (s, br, 1H). Anal. Calcd. for C₂₀H₂₆N₆O₃.0.5 H₂O.: C, 58.95;H, 6.68; N, 20.62; Found: C, 58.83; H, 6.69; N, 20.60.

Preparation of8-[4-(4-fluoro-benzyl)-[1,4]diazepan-1-ylmethyl]-2,9-dihydro-1,2,7,9-tetraaza-phenalen-3-one,25

Synthesized using 1-(4-fluoro-benzyl)-[1,4]diazepane for GeneralProcedure D. 35% overall yield for last two steps. MP: 163-165° C.; MS(ES−): 405; ¹H NMR (400 MHz, CDCl₃): 1.87 (m, 2H), 2.72 (m, 4H), 2.88(m, 4H), 3.63 (s, 2H), 3.65 (s, 2H), 6.99 (t, J=8.4 Hz, 2H), 7.30 (m,3H) 7.61 (s, br, 1H), 7.78 (m, 1H); 7.93 (d, J=7.3 Hz 1H), 10.82 (s, br,1H). Anal. Calcd. for C₂₂H₂₃N₆O. 1.5 H₂O.: C, 60.96; H, 6.05; N, 19.39;Found: C, 61.07; H, 5.97; N, 19.59.

A mesylate salt of 25 was prepared. ¹H NMR (400 MHz, D₂O): 2.06 (m, 2H),2.70 (s, 3H), 3.06 (m, 2H), 3.24 (m, 2H), 3.46 (m, 4H), 3.65 (s, 4H),3.74 (s, 2H), 4.33 (s, 2H), 7.25 (m, 3H), 7.46 (m, 3H), 7.62 (t, J=8.4Hz, 1H). Anal. Calcd. for C₂₂H₂₃FN₆O. 1.3 CH₃SO₃H. 0.5 C₄H₈O₂.2.0 H₂O.:C, 49.70; H, 5.97; N, 13.74; S, 6.82; Found: C, 49.40; H, 5.97; N,13.37; S, 6.65.

Preparation of8-[1,4]diazepan-1-ylmethyl-2,9-dihydro-1,2,7,9-tetraaza-phenalen-3-one,26

Synthesized from compound 24. To a solution of 24 (1.5 g, 3.7 mmol) in30 mL of CH₂Cl₂ was added 6 mL of TFA while stirring at roomtemperature. After 30 minutes, the solvents were evaporated and theresidue was washed with acetonitrile to afford 1.0 g (90%) ofanalytically pure white solid. MP: 147-149° C.; MS (ES−): 297; ¹H NMR(400 MHz, D₂O): 1.96 (m, 2H), 2.82 (t, 2H), 3.01 (t, 2H), 3.28 (t, 4H),3.53 (s, 2H), 7.22 (d, 1H), 7.47 (d, 1H), 7.61 (t, 1H). Anal. Calcd. forC₁₅H₁₈N₆O. 1.1 CF₃CO₂H. 1.0 H₂O.: C, 46.76; H, 4.81; N, 19.02; Found: C,46.64; H, 4.98; N, 19.02.

Preparation of8-[4-(2-trifluoromethyl-benzoyl)-[1,4]diazepan-1-ylmethyl]-2,9-dihydro-1,2,7,9-tetraaza-phenalen-3-one,27

Synthesized from compound 26. To a solution of compound 26 (0.2 g, 0.6mmol) in 5 mL of CH₂Cl₂ was added 1 mmol of TEA and 0.8 mmol of2-trifluoromethyl-benzoyl chloride. The reaction was stirred overnightat room temperature. After the solvents were evaporated, the residue waspurified with semi-preparative HPLC to afford a solid (15% yield). MP:140-142° C.; MS (ES−): 469; ¹H NMR (400 MHz, CDCl₃): 1.92-2.10 (m, 2H),2.91-3.10 (m, 4H), 3.36-3.44 (m, 2H), 3.64-3.74 (m, 2H), 3.93 (m, 2H),7.38 (m, 1H), 7.57 (m, 3H), 7.79 (m, 2H), 7.93 (m, 1H). Anal. Calcd. forC₂₃H₂₁F₃N₆O₂.0.9HCl: C, 54.89; H, 4.39; N, 16.70; Found: C, 54.93; H,4.43; N, 16.34.

Preparation of8-[4-(3-chloro-benzoyl)-[1,4]diazepan-1-ylmethyl]-2,9-dihydro-1,2,7,9-tetraaza-phenalen-3-one,28

Synthesized from compound 26. To a solution of compound 26 (0.2 g, 0.6mmol) in 5 mL of CH₂Cl₂ was added 1 mmol of TEA and 0.8 mmol of3-chloro-benzoyl chloride. The reaction was stirred overnight at roomtemperature. After the solvents were evaporated, the residue waspurified with semi-preparative HPLC to afford a solid (16% yield). MP:147-149° C.; MS (ES−): 436; ¹H NMR (400 MHz, CDCl₃): 1.88-2.08 (m, 2H),2.86-3.07 (m, 4H), 3.52-3.71 (m, 4H), 3.81-3.89 (m, 2H), 7.33-7.43 (m,4H), 7.62 (d, 1H), 7.81 (t, 1H), 7.90 (t, 1H). Anal. Calcd. forC₂₂H₂₁ClN₆O₂.0.7H₂O: C, 54.89; H, 4.39; N, 16.70; Found: C, 54.93; H,4.43; N, 16.34.

Preparation of8-(4-pyridin-2-yl-piperazin-1-ylmethyl)-2,9-dihydro-1,2,7,9-tetraaza-phenalen-3-one,30

Synthesized using 1-pyridin-2-yl-piperazine for General Procedure D. 20%overall yield for last two steps. A mesylate salt of 30 was prepared. MS(ES−): 360; ¹H NMR (400 MHz, DMSO-d₆): 2.37 (s, 6H), 3.52 (s, br, 4H),3.93 (s, br, 4H), 4.30 (s, 2H), 6.93 (t, J=6.6 Hz, 1H), 7.25 (d, J=8.6Hz, 1H), 7.47 (d, J=7.8 Hz, 1H), 7.73 (d, J=7.8 Hz, 1H), 7.82-7.91 (m,2H), 8.16-8.18 (m, 1H), 11.96 (s, 1H). Anal. Calcd. for C₁₉H₁₉N₇O. 1.9CH₃SO₃H. 1.2 H₂O.: C, 44.38; H, 5.17; N, 17.33; S, 10.77; Found: C,44.21; H, 5.19; N, 17.28; S, 10.68.

Preparation of8-{[2-(2-fluoro-phenyl)-ethylamino]-methyl}-2,9-dihydro-1,2,7,9-tetraaza-phenalen-3-one,31

Synthesized using 2-(2-fluoro-phenyl)-ethylamine for General ProcedureD. 20% overall yield for last two steps. A mesylate salt of 31 wasprepared. MS (ES−): 336; ¹H NMR (400 MHz, DMSO-d₆): 2.41 (s, 5H), 3.02(t, J=7.6 Hz, 2H), 3.32 (t, J=8.3 Hz, 2H), 4.16 (s, 2H), 7.19 (t, J=8.8Hz, 2H), 7.32-7.35 (m, 2H), 7.42 (d, J=7.8 Hz, 1H), 7.71 (d, J=7.8 Hz,1H), 7.82 (t, J=8.1 Hz, 1H), 9.10 (s, br, 1H), 11.92 (s, 1H). Anal.Calcd. for C₁₈H₁₆FN₅O. 1.75 CH₃SO₃H. 0.75 H₂O: C, 45.70; H, 4.76; N,13.49; S, 10.81; Found: C, 45.45; H, 4.69; N, 13.42; S, 11.10.

Preparation of8-[4-(4-fluoro-phenyl)-piperazin-1-ylmethyl]-2,9-dihydro-1,2,7,9-tetraaza-phenalen-3-one,32

Synthesized using 4-(4-fluoro-phenyl)-piperazine for General ProcedureD. 57% overall yield for last two steps. A mesylate salt of 32 wasprepared. MS (ES−): 377; ¹H NMR (400 MHz, DMSO-d₆): 2.40 (s, 5H), 3.45(s, br, 4H), 3.59 (s, br, 4H), 4.37 (s, 2H), 7.03-7.15 (m, 4H), 7.44 (d,J=7.8 Hz, 1H), 7.72 (d, J=7.8 Hz, 1H), 7.83 (t, J=7.8 Hz, 1H), 9.8 (s,br, 1H), 11.94 (s, 1H). Anal. Calcd. for C₂₀H₁₉FN₆O. 1.65 CH₃SO₃H: C,46.85; H, 5.01; N, 15.14; S, 9.53; Found: C, 46.74; H, 5.15; N, 15.14;S, 9.53.

Preparation of8-{[2-(4-Fluoro-phenyl)-ethylamino]-methyl}-2,9-dihydro-1,2,7,9-tetraaza-phenalen-3-one,33

Synthesized using 2-(4-fluoro-phenyl)-ethylamine for General ProcedureD. 19% overall yield for last two steps. A mesylate salt of 33 wasprepared. MS (ES−): 336; ¹H NMR (400 MHz, DMSO-d₆): 2.38 (s, 6H),3.06-3.10 (m, 2H), 3.30-3.34 (m, 2H), 4.18 (s, 2H), 7.19-7.22 (m, 2H),7.34-7.42 (m, 3H), 7.71 (d, J=8.6 Hz, 1H), 7.82 (t, J=7.8 Hz, 1H), 9.6(s, br, 1H), 11.92 (s, 1H). Anal. Calcd. for C₁₈H₁₆FN₅O. 2.0 CH₃SO₃H: C,45.36; H, 4.57; N, 13.22; S, 12.11; Found: C, 45.34; H, 4.58; N, 13.16;S, 11.88.

Preparation of8-(4-acetyl-[1,4]diazepan-1-ylmethyl)-2,9-dihydro-1,2,7,9-tetraaza-phenalen-3-one,34

Synthesized using [1,4]diazepane-1-yl-ethanone for General Procedure D.16% overall yield for last two steps. MP: 191-193° C.; MS (ES−): 339; ¹HNMR (400 MHz, CDCl₃): 2.11 (s, 3H), 2.84-2.93 (m, 4H), 3.56-3.76 (m,6H), 7.66 (m, 1H), 7.83-7.92 (m, 2H), 9.3 (s, br, 1H), 11.3 (s, br, 1H).Anal. Calcd. for C₁₇H₂₀N₆O₂.0.6H₂O: C, 58.14; H, 6.08; N, 23.93; Found:C, 58.09; H, 6.18; N, 24.08.

Preparation of8-(phenethylamino-methyl)-2,9-dihydro-1,2,7,9-tetraaza-phenalen-3-one,35

Synthesized using phenetyhlamine for General Procedure D. 29% overallyield for last two steps. A mesylate salt of 35 was prepared. MS (ES−):358; ¹H NMR (400 MHz, DMSO-d₆): 2.32 (s, 3H), 3.00-3.04 (m, 2H),3.31-3.36 (m, 2H), 4.15 (s, 1H), 7.27-7.42 (m, 6H), 7.71 (d, J=7.8 Hz,1H), 7.82 (t, J=7.8 Hz, 1H), 9.70 (s, br, 1H), 11.92 (s, 1H). Anal.Calcd. for C₁₈H₁₇N₅O. 1.0 CH₃SO₃H. 1.8 H₂O: C, 50.95; H, 5.54; N, 15.64;S, 7.16; Found: C, 50.95; H, 5.54; N, 15.64; S, 7.16;

Preparation of8-(4-phenyl-piperidin-1-ylmethyl)-2,9-dihydro-1,2,7,9-tetraaza-phenalen-3-one,36

Synthesized using 4-phenyl-piperidine for General Procedure D. 33%overall yield for last two steps. MS (ES−): 318; ¹H NMR (400 MHz,DMSO-d₆): 1.87-1.93 (m, 4H), 2.37-2.46 (m, 2H), 2.56 (m, 1H), 3.10-3.14(m, 2H), 3.54 (s, 2H), 7.17-7.34 (m, 5H), 7.56 (bs, 1H), 7.76 (t, J=7.8Hz, 1H), 7.93 (d, J=7.8 Hz, 1H), 11.10 (s, br, 1H), 11.76 (s, 1H). Amesylate salt of 36 was prepared. ¹H NMR (400 MHz, D₂O): 2.08 (m, 4H),2.95 (m, 1H), 3.34 (m, 2H), 3.84 (m, 2H), 4.23 (s, 2H), 7.21-7.39 (m,6H), 7.59 (m, 1H), 7.70 (m, 1H). Anal. Calcd. for C₂₁H₂₁N₅O. 1.3CH₃SO₃H. 0.5 H₂O: C, 54.29; H, 5.56; N, 14.19; S, 8.45; Found: C, 54.03;H, 5.65; N, 13.98; S, 8.64.

Preparation of8-(1,3-dihydro-isoindol-2-ylmethyl)-2,9-dihydro-1,2,7,9-tetraaza-phenalen-3-one,37

Synthesized using isoindoline for General Procedure D. 40% overall yieldfor last two steps. MS (ES−): 316; ¹H NMR (400 MHz, DMSO-d₆): 3.77 (s,2H), 4.04 (s, 4H), 7.20-7.30 (m, 4H), 7.49 (d, J=7.8 Hz, 1H), 7.6 (d,J=7.8 Hz, 1H), 7.74 (t, J=7.8 Hz, 1H), 11.34 (s, br, 1H), 11.78 (s, 1H).A mesylate salt of 37 was prepared. ¹H NMR (400 MHz, DMSO-d₆): 2.34 (s,3H), 4.64 (s, 2H), 4.87 (s, 4H), 7.39-7.46 (m, 5H), 7.72 (d, J=7.8 Hz,1H), 7.83 (t, J=8.1 Hz, 1H), 11.30 (s, br, 1H), 11.95 (s, 1H). Anal.Calcd. for C₁₈H₁₅N₅O. 1.25 CH₃SO₃H. 2.0 H₂O: C, 48.83; H, 5.11; N,14.79; S, 8.46; Found: C, 48.80; H, 5.11; N, 14.97; S 8.71.

Preparation of8-(4-benzenesulfonyl-[1,4]diazepan-1-ylmethyl)-2,9-dihydro-1,2,7,9-tetraaza-phenalen-3-one,38

Synthesized from compound 26. To a solution of 26 (0.2 g, 0.67 mmol) in5 mL of CH₂Cl₂ was added TEA (2 mmol) and benzensulfonyl chloride (1mmol). The mixture was stirred at room temperature over night. After thesolvents were evaporated, the residue was poured into 10 mL of H₂O andthe product was purified by preparative HPLC to afford analytically purewhite solid (5% yield). MP: 265-268° C.; MS (ES−): 437; ¹H NMR (400 MHz,DMSO-d₆): 1.79 (m, 2H), 2.50 (m, 4H), 2.79 (m, 4H), 3.51 (s, 2H), 7.44(d, 1H), 7.62-7.79 (m, 7H), 11.1 (s, br, 1H), 11.75 (s, 1H). Anal.Calcd. for C₂₁H₂₂N₆O₃S. 0.5 H₂O: C, 56.36; H, 5.18; N, 18.78; S, 7.17;Found: C, 56.44; H, 5.12; N, 19.00; S, 7.19.

A mesylate salt of 38 was prepared. MS (ES+): 439; ¹H NMR (400 MHz,D₂O): 2.18 (m, 2H), 2.35 (s, 6H), 3.36 (m, 2H), 3.65 (m, 6H), 4.3 (s,2H), 7.24 (d, 1H), 7.51-7.71 (m, 7H). Anal. Calcd. for C₂₁H₂₂N₆O₃S. 1.8CH₃SO₃H. 1.0 H₂O: C, 43.50; H, 5.00; N, 13.35; S, 14.26; Found: C,43.61; H, 5.00; N, 13.15; S, 14.59.

Preparation of8-(4-pyridin-4-yl-piperazin-1-ylmethyl)-2,9-dihydro-1,2,7,9-tetraaza-phenalen-3-one,39

Synthesized using 1-(4-pyridyl)piperazine for General Procedure D. 10%overall yield for last two steps. MS (ES−): 360; ¹H NMR (400 MHz,DMSO-d₆): 2.80 (t, J=5.0 Hz, 4H), 3.61 (t, J=5.0 Hz, 4H), 3.99 (s, 2H),6.83 (d, J=7.1 Hz, 2H), 7.42-7.45 (m, 1H), 7.73-7.81 (m, 2H), 8.26 (d,J=7.1 Hz, 2H), 11.20 (s, br, 1H), 11.90 (s, 1H). An HCl salt of 39 wasprepared. ¹H NMR (400 MHz, D₂O): 2.74-2.77 (m, 4H), 3.43 (s, 2H),3.35-3.69 (m, 4H), 6.93 (d, J=7.1 Hz, 2H), 7.13 (d, J=8.0 Hz, 1H), 7.37(d, J=7.8 Hz, 1H), 7.58 (t, J=7.8 Hz, 1H), 7.92 (d, J=7.1 Hz, 2H). Anal.Calcd. for C₁₉H₁₉N₇O. 1.0 HCl. 2.5H₂O: C, 51.53; H, 5.69; N, 22.14; Cl,8.00; Found: C, 51.46; H, 5.69; N, 21.90; Cl, 8.27.

Preparation of8-(4-benzyl-piperazin-1-ylmethyl)-2,9-dihydro-1,2,7,9-tetraaza-phenalen-3-one,40

Synthesized using 4-benzyl-piperazine for General Procedure D. 12%overall yield for last two steps. MS (ES−): 373; ¹H NMR (400 MHz,DMSO-d₆): 2.44 (s, br, 4H), 3.35 (s, br, 4H), 3.48 (s, 2H), 7.23-7.34(m, 5H), 7.49 (d, J=8.8 Hz, 1H), 7.62 (d, J=7.8 Hz, 1H), 7.74 (t, J=7.8Hz, 1H), 11.10 (s, br, 1H), 11.77 (s, 1H). An HCl salt of 40 wasprepared. ¹H NMR (400 MHz, D₂O): 2.54-2.70 (m, 2H), 3.10-3.50 (m, 6H),3.48 (s, 2H), 4.35 (s, 2H), 7.20 (d, J=8.1 Hz, 1H), 7.45 (t, J=7.8 Hz,1H), 7.47-7.51 (m, 5H), 7.62 (t, J=8.1 Hz, 1H). Anal. Calcd. forC₂₁H₂₂N₆O. 1.0 HCl. 2.5H₂O: C, 55.32; H, 6.19; N, 18.43; Found: C,55.54; H, 6.08; N, 18.32.

Preparation of8-(4-methyl-[1,4]diazepan-1-ylmethyl)-2,9-dihydro-1,2,7,9-tetraaza-phenalen-3-one,41

Synthesized using 1-methyl-[1,4]diazepane for General Procedure D. 24%overall yield for last two steps. MS (ES−): 311; ¹H NMR (400 MHz,DMSO-d₆): 1.75 (m, 2H), 2.26 (s, 3H), 2.55 (m, 4H), 2.79 (m, 4H), 3.48(s, 2H), 7.52 (d, J=8.2 Hz, 1H), 7.64 (d, J=7.2 Hz, 1H), 7.75 (t, J=8.1Hz, 1H), 11.55 (s, 1H). Anal. Calcd. for C₁₆H₂₀N₆O. 0.95 H₂O: C, 58.33;H, 6.70; N, 25.51; Found: C, 58.32; H, 6.65; N, 25.53.

Preparation of8-[4-(1H-indol-3-yl)-piperidin-1-ylmethyl]-2,9-dihydro-1,2,7,9-tetraaza-phenalen-3-one,42

Synthesized using 3-piperidin-4-yl-1H-indole for General Procedure D.19% overall yield for last two steps. MS (ES−): 397; ¹H NMR (400 MHz,DMSO-d₆): 1.83-1.94 (m, 4H), 2.31 (m, 2H), 2.50 (s, 2H), 2.79-2.99 (m,3H), 6.96-7.09 (m, 3H), 7.32 (d, J=8.1 Hz, 1H), 7.54-7.63 (m, 3H), 7.75(t, J=7.3 Hz, 1H), 10.79 (s, 1H), 11.80 (s, 1H). A mesylate salt of 42was prepared. ¹H NMR (400 MHz, DMSO-d₆): 2.15 (m, 4H), 2.32 (s, 3H),3.11 (m, 1H), 3.52 (m, 2H), 3.73 (m, 2H), 4.29 (s, 2H), 7.10-7.18 (m,3H), 7.36 (d, 1H); 7.46 (d, J=8.2 Hz, 1H), 7.69-7.83 (m, 3H), 10.91 (s,1H), 11.93 (s, 1H). Anal. Calcd. for C₂₃H₂₂N₆O. 1.0 CH₃SO₃H. 1.25H₂O: C,55.23; H, 5.62; N, 16.91; S, 6.14; Found: C, 55.27; H, 5.53; N, 16.95;S, 6.00.

Preparation of8-[(2-pyridin-4-yl-ethylamino)-methyl]-2,9-dihydro-1,2,7,9-tetraaza-phenalen-3-one,43

Synthesized using 4-ethylamino-pyridine for General Procedure D. 10%overall yield for last two steps. An HCl salt of 43 was prepared. MS(ES−): 319; ¹H NMR (400 MHz, D₂O): 3.28 (t, J=7.8 Hz, 2H), 3.53 (t,J=7.8 Hz, 2H), 4.09 (s, 2H), 7.02 (d, J=8.0 Hz, 1H), 7.35 (d, J=8.0 Hz,1H), 7.52 (t, J=8.0 Hz, 1H), 7.70 (d, J=5.3 Hz, 2H), 8.52 (d, J=5.3 Hz,2H). Anal. Calcd. for C₁₇H₁₆N₆O. 1.3 HCl. 2.6 H₂O. 0.1 N₂H₄: C, 47.52;H, 5.38; N, 20.27; Found: C, 47.12; H, 5.26; N, 20.67.

Preparation of8-(3,4-dihydro-1H-isoquinolin-2-ylmethyl)-2,9-dihydro-1,2,7,9-tetraaza-phenalen-3-one,44

Synthesized using 1,2,3,4-tetrahydro-isoquinoline for General ProcedureD. 30% overall yield for last two steps. MS (ES−): 330; ¹H NMR (400 MHz,DMSO-d₆): 2.81-2.90 (m, 4H); 3.52 (s, 2H), 3.72 (s, 2H), 7.05-7.25 (m,4H), 7.51 (d, J=7.8 Hz, 1H), 7.63 (d, J=8.0 Hz, 1H), 7.74 (t, J=8.0 Hz,1H), 11.30 (s, br, 1H), 11.91 (s, 1H). Anal. Calcd. for C₁₉H₁₇N₅O: C,68.87; H, 5.17; N, 21.13; Found: C, 68.34; H, 5.19; N, 21.30.

A mesylate salt of 44 was prepared. MS (ES−): 330; ¹H NMR (400 MHz,D₂O): 2.80 (s, 3H), 3.31 (t, 2H), 3.85 (m, 2H), 4.47 (s, 2H), 4.68 (s,2H), 7.23 (d, J=7.8 Hz, 1H), 7.28-7.42 (m, 4H), 7.67 (d, J=8.0 Hz, 1H);7.80 (t, J=7.9 Hz, 1H). Anal. Calcd. for C₁₉H₁₇N₅O. 1.12 CH₃SO₃H. 2.0H₂O: C, 50.87; H, 5.41; N, 14.74; S, 7.56; Found: C, 50.89; H, 5.47; N,14.84; S, 7.63.

Preparation of8-(5,6-Dimethoxy-3,4-dihydro-1H-isoquinolin-2-ylmethyl)-2,9-dihydro-1,2,7,9-tetraaza-phenalen-3-one,45

Synthesized using 5,6-dimethoxy-1,2,3,4-tetrahydro-isoquinoline forGeneral Procedure D. 29% overall yield for last two steps. MS (ES−):311; ¹H NMR (400 MHz, DMSO-d₆): 2.79 (s, 4H), 3.49 (s, 2H), 3.61 (s,2H), 3.67 (s, 3H), 3.70 (s, 3H), 6.69 (d, J=8.8 Hz, 2H), 7.48 (d, J=7.6Hz, 1H), 7.63 (d, J=7.8 Hz, 1H), 7.74 (t, J=7.6 Hz, 1H), 11.55 (s, 1H).Anal. Calcd. for C₂₁H₂₁N₅O₃: C, 64.44; H, 5.41; N, 17.89; Found: C,64.24; H, 5.43; N, 17.98.

A mesylate salt of 45 was prepared. MS (ES−): 330; ¹H NMR (400 MHz,D₂O): 2.82 (s, 3H), 3.21 (t, 2H), 3.65-3.85 (m, 8H), 4.48 (s, 2H), 4.60(s, 2H), 6.75 (s, 1H), 6.83 (s, 1H), 7.38 (d, 1H), 7.71 (d, 1H), 7.82(t, 1H). Anal. Calcd. for C₂₁H₂₁N₅O₃.1.18CH₃SO₃H. 1.75 H₂O: C, 49.70; H,5.49; N, 13.07; S, 7.03; Found: C, 49.77; H, 5.49; N, 13.17; S, 7.03.

Preparation of8-[4-(3-Trifluoromethyl-benzenesulfonyl)-[1,4]diazepan-1-ylmethyl]-2,9-dihydro-1,2,7,9-tetraaza-phenalen-3-one,46

Synthesized from compound 26. To a solution of 26 (0.2 g, 0.67 mmol) in5 mL of CH₂Cl₂ was added TEA (2 mmol) and3-trifluoromethyl-benzenesulfony chloride (1 mmol). The mixture wasstirred at room temperature over night. After the solvents wereevaporated, the residue was poured into 10 mL of H₂O and the product waspurified by preparative HPLC to afford analytically pure white solid(15% yield). MS (ES+): 507; ¹H NMR (400 MHz, DMSO-d₆): 1.82 (m, 2H),2.73-2.81 (m, 4H), 3.25-3.42 (m, 6H), 7.44 (d, J=7.8 Hz, 1H), 7.63 (d,J=7.2 Hz, 1H), 7.74 (t, J=7.8 Hz, 1H), 7.89 (t, J=8.2 Hz, 1H), 8.04-8.13(m, 3H), 11.10 (s, br, 1H), 11.75 (s, 1H). Anal. Calcd. forC₂₂H₂₁F₃N₆O₃S. 1.1 H₂O: C, 50.21; H, 4.44; N, 15.97; S, 6.09; Found: C,50.19; H, 4.54; N, 15.50; S, 5.97.

General Procedure F: Preparation of Compounds 47A and 47B:

Displacement of the chloro group of compound 4 with piperazine or[1,4]diazepane using General procedure F provides the compound 47A or47B. To a stirring solution of 4 (1 eq) in acetonitrile was addedpiperazine or [1,4]diazepane (large excess) under a blanket of nitrogen.The solution was allowed to stir overnight and then evaporated todryness. The crude material was purified via silica plug with 9:1dichloromethane:methanol to afford a white solid,4-Oxo-2-piperazin-1-ylmethyl-3,4-dihydro-quinazoline-5-carboxylic acidmethyl ester, 47A or2-[1,4]diazepan-1-ylmethyl-4-oxo-3,4-dihydro-quinazoline-5-carboxylicacid methyl ester, 47B.

General Procedure G: Preparation of Compounds 48A and 48B:

A reaction of amine 47A or 47B with various sulfonyl chloride yieldssulfonyl amide 48A or 48B. To a stirring solution of 47A or 47B (1.0 eq)in pyridine was added various sulfonyl chloride (1.1 eq). The reactionwas allowed to stir overnight and then was evaporated to dryness. Theresidue was then extracted with dichloromethane and washed with brine.The product was evaporated to dryness and used without furtherpurification.

General Procedure E: Preparation of compounds 49A and 49B:

A 2,9-dihydro-1,2,7,9-tetraaza-phenalen-3-one ring can be formed bycondensation of the compound 48A or 48B with hydrazine. To a solution ofthe compounds 6 in absolute ethanol is added excess anhydrous hydrazineat room temperature. The solution is refluxed for overnight and cooledto room temperature. Ice-cold water is added and white solid isseparated. The solid is collected by vacuum filtration and washed withwater and small amount of methanol to give white solid products 6 in40-90% of yield. An example was given in the preparation of compounds49A and 49B.

Example 2 Preparation of8-[4-(4-methoxy-benzenesulfonyl)-piperazin-1-ylmethyl]-2,9-dihydro-1,2,7,9-tetraaza-phenalen-3-one,50

To a stirring solution of 4 (2.2 g, 8.73 mmol, 1 eq) in 200 mL ofacetonitrile was added piperazine (14 g, 0.162 mol, large excess) undera blanket of nitrogen. The solution was allowed to stir overnight andthen evaporated to dryness. The crude material was purified via silicaplug with 9:1 dichloromethane:methanol to afford 2.0 g of a fluffy whitesolid, 4-Oxo-2-piperazin-1-ylmethyl-3,4-dihydro-quinazoline-5-carboxylicacid methyl ester, 47A. MS (ES−): 301; ¹H NMR (400 MHz, DMSO-d₆):2.40-2.43 (m, 4H), 2.69-2.72 (m, 4H), 3.41 (s, 2H), 3.83 (s, 3H), 7.44(d, J=7.2 Hz, 1H), 7.74 (d, J=8.2 Hz, 1H), 7.82 (t, J=7.8 Hz, 1H).

To a stirring solution of 47A (170 mg, 0.56 mmol, 1 eq) in 5 mL ofpyridine was added 4-methoxybenzene sulfonyl chloride (130 mg, 0.62mmol, 1.1 eq) resulting in a bright yellow solution. The reaction wasallowed to stir overnight and then was evaporated to dryness. The waxyresidue was then extracted with dichloromethane and washed with brine.The crude material was dissolved in 10 mL of EtOH and 5 mL of hydrazinemonohydrate (large excess). This solution was refluxed overnightresulting in a heavy white precipitate which was filtered, washed withethyl ether and dried to give an off white solid. This solid was thenpurified via chromatography to afford 112 mg of analytically purecompound 50. A mesylate salt of 50 was prepared. 8% overall yield forlast three steps. MS (ES+): 455; ¹H NMR (400 MHz, DMSO-d₆): 2.34 (s,3H), 3.19 (bs, 4H), 3.44 (bs, 4H), 3.89 (s, 3H), 4.20 (s, 2H), 7.25 (d,J=9.0 Hz, 2H), 7.72 (d, J=7.8 Hz, 1H), 7.70-7.83 (m, 4H), 11.20 (s, br,1H), 11.93 (s, 1H). Anal. Calcd. for C₂₁H₂₂N₆O₄S. 1.5 CH₃SO₃H. 3.0 H₂O.1.0 N₂H₄: C, 41.20; H, 5.29; N, 13.24; S, 12.22; Found: C, 41.07; H,5.09; N, 13.53; S, 12.62.

The following compounds were synthesized from the similar procedures ofpreparation of compound 50, using the appropriate corresponding sulfonylchloride.

Preparation of8-[4-(3-fluoro-benzenesulfonyl)-piperazin-1-ylmethyl]-2,9-dihydro-1,2,7,9-tetraaza-phenalen-3-one,51

Synthesized using 3-fluoro-benzenesulfonyl chloride and compound 47A forGeneral Procedure G. A mesylate salt of 51 was prepared. 35% overallyield for last three steps. MS (ES−): 441; ¹H NMR (400 MHz, DMSO-d₆):2.31 (s, 3H), 3.25 (bs, 4H), 3.39 (bs, 4H), 4.15 (s, 2H), 7.42 (d, J=7.8Hz, 1H), 7.65-7.71 (m, 4H), 7.78-7.82 (m, 2H), 11.78 (s, 1H). Anal.Calcd. for C₂₀H₁₉N₆O₃S. 1.25 CH₃SO₃H. 2.4 H₂O: C, 42.43; H, 4.87; N,13.87; S, 11.91; Found: C, 42.13; H, 4.79; N, 13.48; S, 11.89.

Preparation of8-[4-(toluene-4-sulfonyl)-piperazin-1-ylmethyl]-2,9-dihydro-1,2,7,9-tetraaza-phenalen-3-one,52

Synthesized using toluene-4-sulfonyl chloride and compound 47A forGeneral Procedure G. A mesylate salt of 52 was prepared. 38% overallyield for last three steps. MS (ES−): 438; ¹H NMR (400 MHz, DMSO-d₆):2.36 (s, 3H), 2.45 (s, 3H), 3.20 (bs, 4H), 3.46 (bs, 4H), 4.22 (s, 2H),7.43 (d, J=7.8 Hz, 1H), 7.54 (d, J=7.8 Hz, 2H), 7.68-7.81 (m, 4H), 11.90(s, 1H). Anal. Calcd. for C₂₁H₂₂N₆O₃S. 1.3 CH₃SO₃H. 4.0 H₂O: C, 42.25;H, 5.58; N, 13.22; S, 11.61; Found: C, 42.63; H, 5.53; N, 13.40; S,11.90.

Preparation of8-(4-benzenesulfonyl-piperazin-1-ylmethyl)-2,9-dihydro-1,2,7,9-tetraaza-phenalen-3-one,53

Synthesized using benzensulfonyl chloride and compound 47A for GeneralProcedure G. A mesylate salt of 53 was prepared. 30% overall yield forlast three steps. MS (ES−): 438; ¹H NMR (400 MHz, DMSO-d₆): 2.70 (s,3H), 3.36 (bs, 4H), 3.51 (bs, 4H), 4.14 (s, 2H), 7.11 (d, J=8.0 Hz, 1H),7.40-7.70 (m, 7H), 11.90 (s, 1H). Anal. Calcd. for C₂₀H₂₀N₆O₃S. 1.2CH₃SO₃H. 2.5H₂O. 0.08 N₂H₄: C, 43.36; H, 5.17; N, 14.67; S, 12.01;Found: C, 43.00; H, 5.17; N, 15.05; S, 12.40.

Preparation of8-[4-(3-trifluoromethyl-benzenesulfonyl)-piperazin-1-ylmethyl]-2,9-dihydro-1,2,7,9-tetraaza-phenalen-3-one,54

Synthesized using 3-trifluoro-benzensulfonyl chloride and compound 47Afor General Procedure G. A mesylate salt of 54 was prepared. 15% overallyield for last three steps. MS (ES−): 438; ¹H NMR (400 MHz, DMSO-d₆):2.32 (s, 3H), 3.26-3.35 (m 8H), 4.10 (s, 2H), 7.43 (d, J=8.0 Hz, 1H),7.69-7.80 (m, 2H), 7.98-8.26 (m, 4H), 11.92 (s, 1H)

Anal. Calcd. for C₂₁H₁₉F₃N₆O₃S. 1.3 CH₃SO₃H. 2.0 H₂O: C, 40.99; H, 4.35;N, 12.86; S, 11.29; Found: C, 40.71; H, 4.60; N, 12.68; S, 11.50.

Preparation of8-[4-(4-chloro-benzenesulfonyl)-piperazin-1-ylmethyl]-2,9-dihydro-1,2,7,9-tetraaza-phenalen-3-one,55

Synthesized using 4-chlorobenzensulfonyl chloride and compound 47A forGeneral Procedure G. A mesylate salt of 55 was prepared. 15% overallyield for last three steps. MS (ES−): 458; ¹H NMR (400 MHz, DMSO-d₆):2.31 (s, 3H), 3.18 (bs, 4H), 3.40 (bs, 4H), 3.98 (s, 2H), 7.43 (d, J=7.7Hz, 1H), 7.68-7.84 (m, 5H), 11.90 (s, 1H).

Anal. Calcd. for C₂₀H₁₉ClN₆O₃S. 1.3 CH₃SO₃H. 2.0 H₂O: C, 41.27; H, 4.59;N, 13.56; S, 11.90; Found: C, 41.07; H, 4.66; N, 13.30; S, 11.89.

Preparation of8-[4-(4-fluoro-benzenesulfonyl)-piperazin-1-ylmethyl]-2,9-dihydro-1,2,7,9-tetraaza-phenalen-3-one,56

Synthesized using 4-fluorobenzensulfonyl chloride and compound 47A forGeneral Procedure G. An HCl salt of 56 was prepared. 42% overall yieldfor last three steps. MS (ES−): 441; ¹H NMR (400 MHz, DMSO-d₆): 2.31 (s,3H), 3.18 (bs, 4H), 3.40 (bs, 4H), 3.98 (s, 2H), 7.43 (d, J=7.8 Hz, 1H),7.68-7.84 (m, 5H), 11.90 (s, 1H).

Preparation of8-[4-(4-isopropyl-benzenesulfonyl)-piperazin-1-ylmethyl]-2,9-dihydro-1,2,7,9-tetraaza-phenalen-3-one,57

Synthesized using 4-isopropylbenzensulfonyl chloride and compound 47Afor General Procedure G. A mesylate salt of 57 was prepared. 22% overallyield for last three steps. MS (ES−): 465; ¹H NMR (400 MHz, DMSO-d₆):1.26 (d, J=6.8 Hz, 6H), 2.33 (s, 3H), 3.01-3.05 (m, 1H), 3.16-3.32 (m,10H), 7.41 (d, J=8.1 Hz, 1H), 7.59 (d, J=8.6 Hz, 2H), 7.68-7.80 (m, 4H),11.89 (s, 1H). Anal. Calcd. for C₂₃H₂₆N₆O₃S. 1.35 CH₃SO₃H. 1.75 H₂O. 0.1N₂H₄: C, 46.35; H, 5.64; N, 13.76; S, 11.94; Found: C, 46.01; H, 5.62;N, 13.80; S, 12.33.

Preparation of8-[4-(4-tert-butyl-benzenesulfonyl)-piperazin-1-ylmethyl]-2,9-dihydro-1,2,7,9-tetraaza-phenalen-3-one,58

Synthesized using 4-tertbutylbenzensulfonyl chloride and compound 47Afor General Procedure G. A mesylate salt of 58 was prepared. 23% overallyield for last three steps. MS (ES−): 480; ¹H NMR (400 MHz, DMSO-d₆):1.25 (s, 9H), 2.21 (s, 3H), 3.05-3.15 (m, 8H), 3.99 (bs, 2H), 7.32 (d,J=8.1 Hz, 1H), 7.59-7.72 (m, 6H), 11.81 (s, 1H). Anal. Calcd. forC₂₄H₂₈N₆O₃S. 1.5 CH₃SO₃H. 2.75 H₂O: C, 45.42; H, 5.90; N, 12.46; S,11.89; Found: C, 45.23; H, 5.76; N, 12.84; S, 12.17.

Preparation of8-[4-(4-isopropyl-benzenesulfonyl)-[1,4]diazepan-1-ylmethyl]-2,9-dihydro-1,2,7,9-tetraaza-phenalen-3-one,59

Synthesized using 4-isopropylbenzensulfonyl chloride and compound 47Bfor General Procedure G. 22% overall yield for last two steps. MS (ES−):479; ¹H NMR (400 MHz, DMSO-d₆): 1.23 (d, 6H), 1.79 (m, 2H), 2.40-2.55(m, 4H), 2.71-2.90 (n 4H), 3.00 (m, 1H), 3.48 (s, 2H), 7.48 (m, 3H),7.73 (m, 4H), 11.80 (s, 1H). Anal. Calcd. for C₂₄H₂₈N₆O₃S: C, 59.98; H,5.87; N, 17.49; S, 6.67; Found: C, 60.02; H, 5.85; N, 17.55; S, 6.52.

Preparation of8-[4-(4-chloro-benzenesulfonyl)-[1,4]diazepan-1-ylmethyl]-2,9-dihydro-1,2,7,9-tetraaza-phenalen-3-one,60

Synthesized using 4-chloro-benzenesulfony chloride and compound 47B forGeneral Procedure G. 8% overall yield for last three steps. MS (ES−):472; ¹H NMR (400 MHz, DMSO-d₆): 1.80 (m, 2H), 2.73-2.78 (m, 4H), 3.50(m, 4H), 3.69 (s, 2H), 7.45 (d, J=8.2 Hz, 1H), 7.71-7.83 (m, 6H), 10.95(s, br, 1H), 11.76 (s, 1H). A mesylate salt of 60 was prepared. ¹H NMR(400 MHz, D₂O): 1.92 (m, 2H), 2.73 (s, 5H), 3.50-3.77 (m, 8H), 4.36 (s,2H), 7.49 (d, J=7.2 Hz, 1H), 7.75 (t, J=8.1 Hz, 2H), 7.78-7.93 (m 4H).Anal. Calcd. for C₂₁H₂₁ClN₆O₃S. 1.61 CH₃SO₃H: C, 39.57; H, 4.99; N,12.25; S, 12.20; Found: C, 39.50; H, 5.29; N, 12.57; S, 12.47.

Preparation of8-[4-(3-fluoro-benzenesulfonyl)-[1,4]diazepan-1-ylmethyl]-2,9-dihydro-1,2,7,9-tetraaza-phenalen-3-one,61

Synthesized using 3-fluoro-benzenesulfony chloride and compound 47B forGeneral Procedure G. 16% overall yield for last two steps. MS (ES+):457; ¹H NMR (400 MHz, DMSO-d₆): 1.79 (m, 2H), 2.70-2.81 (m, 4H),3.26-3.40 (m, 4H), 3.48 (s, 2H), 7.45 (d, J=7.3 Hz, 1H); 7.55-7.74 (m,6H), 11.10 (s, br, 1H), 11.75 (s, 1H). Anal. Calcd. for C₂₁H₂₁FN₆O₃S.1.15 H₂O: C, 52.85; H, 4.92; N, 17.61; S, 6.72; Found: C, 52.88; H,4.93; N, 17.43; S, 6.48.

Preparation of8-[4-(4-methoxy-benzenesulfonyl)-1-[1,4]diazepan-1-ylmethyl]-2,9-dihydro-1,2,7,9-tetraaza-phenalen-3-one,62

Synthesized using 4-methoxy-benzensulfonyl chloride and compound 47B forGeneral Procedure G. 21% overall yield for last two steps. MS (ES+):469; ¹H NMR (400 MHz, DMSO-d₆): 1.78 (m, 2H), 2.72-2.79 (m, 4H),3.30-3.39 (m, 4H), 3.48 (s, 2H), 3.84 (s, 3H), 7.14 (d, J=8.2 Hz, 2H),7.48 (d, J=8.1 Hz, 1H), 7.63 (d, J=7.2 Hz, 1H); 7.09-7.22 (m, 3H), 11.10(s, br, 1H), 11.80 (s, 1H). Anal. Calcd. for C₂₂H₂₄N₆O₄S. 1.0 H₂O: C,54.31; H, 5.39; N, 17.27; S, 6.59; Found: C, 54.38; H, 5.34; N, 17.28;S, 6.19.

Preparation of8-[4-(4-tert-butyl-benzenesulfonyl)-[1,4]diazepan-1-ylmethyl]-2,9-dihydro-1,2,7,9-tetraaza-phenalen-3-one,63

Synthesized using 4-t-butyl-benzenesulfony chloride and compound 47B forGeneral Procedure G. 14% overall yield for last two steps. MS (ES−):493; ¹H NMR (400 MHz, DMSO-d₆): 1.31 (s, 9H), 1.79 (m, 2H), 2.73-2.86(m, 4H), 3.26-3.41 (m, 4H), 3.48 (s, 2H), 7.45 (d, J=8.6 Hz, 1H),7.62-7.76 (m, 6H), 11.20 (s, br, 1H), 11.80 (s, 1H). Anal. Calcd. forC₂₅H₃₀N₆O₃S: C, 60.71; H, 6.11; N, 16.99; S, 6.48; Found: C, 60.78; H,6.10; N, 17.08; S, 6.36.

Preparation of8-[4-(4-amino-benzenesulfonyl)-[1,4]diazepan-1-ylmethyl]-2,9-dihydro-1,2,7,9-tetraaza-phenalen-3-one,64

Synthesized using 4-nitro-benzenesulfony chloride and compound 47B forGeneral Procedure G. 14% overall yield for last two steps. MS (ES−):452; ¹H NMR (400 MHz, DMSO-d₆): 1.76 (m, 2H), 2.71-2.79 (m, 4H),3.21-3.31 (m, 4H), 3.46 (s, 2H), 6.01 (s, 2H), 6.64 (d, J=8.6 Hz, 2H),7.39 (d, J=8.6 Hz, 2H), 7.48 (d, J=8.0 Hz, 1H), 7.63 (d, J=7.9 Hz, 1H),7.74 (t, J=7.8 Hz, 1H), 11.10 (s, br, 1H), 11.75 (s, 1H). Anal. Calcd.for C₂₁H₂₃N₇O₃S. 0.5 H₂O: C, 54.53; H, 5.23; N, 21.20; S, 6.93; Found:C, 54.50; H, 5.24; N, 20.84; S, 6.74.

Preparation of8-[4-(biphenyl-4-sulfonyl)-[1,4]diazepan-1-ylmethyl]-2,9-dihydro-1,2,7,9-tetraaza-phenalen-3-one,65

Synthesized using biphenyl-4-sulfony chloride and compound 47B forGeneral Procedure G. 10% overall yield for last two steps. MS (ES−):513; ¹H NMR (400 MHz, DMSO-d₆): 1.82 (m, 2H), 2.73-2.83 (m, 4H),3.29-3.41 (m, 4H), 3.48 (s, 2H), 7.47-7.53 (m, 4H), 7.62 (d, J=8.1 Hz,1H), 7.68-7.78 (m, 3H), 7.82-7.93 (m, 4H), 11.00 (s, br, 1H), 11.75 (s,1H). Anal. Calcd. for C₂₇H₂₆N₆O₃S. 2.3H₂O: C, 58.32; H, 5.55; N, 15.11;S, 5.77; Found: C, 58.24; H, 4.89; N, 15.10; S, 5.79.

Preparation of8-[4-(4-amino-benzenesulfonyl)-piperazin-1-ylmethyl]-2,9-dihydro-1,2,7,9-tetraaza-phenalen-3-one,66

Synthesized using 4-nitrobenzene sulfonyl chloride and compound 47A forGeneral Procedure G. A mesylate salt of 66 was prepared. 35% overallyield for last three steps. MS (ES−): 438; ¹H NMR (400 MHz, DMSO-d₆):2.32 (s, 3H), 3.13 (bs, 4H), 3.42 (bs, 4H), 4.18 (s, 2H), 6.71 (d, J=8.8Hz, 2H), 7.40-7.43 (m, 3H), 7.70-7.80 (m, 2H), 11.20 (s, br, 1H), 11.92(s, 1H). Anal. Calcd. for C₂₀H₂₁N₇O₃S. 1.3 CH₃SO₃H. 2.75 H₂O: C, 41.67;H, 5.20; N, 15.97; S, 12.01; Found: C, 41.76; H, 5.25; N, 15.92; S,12.22.

General Procedure L to prepare compounds 71. To a stirring solution of70 (1.0 eq) in THF under nitrogen was added TEA (1 mL, excess) andeither sulfonyl chloride or acid chloride (1.2 eq). The reaction wasallowed to stir for four hours after which time it was evaporated andextracted with CH₂Cl₂/H₂O, dried and condensed. Crude material wasfurther purified via column chromatography using 9:1 CH₂Cl₂₁MeOH toafford analytically pure products 71.

Example 3 Preparation of(3-oxo-2,9-dihydro-3H-1,2,7,9-tetraaza-phenalen-8-ylmethyl)-carbamicAcid tert-butyl Ester, 69

Procedure H to prepare2-aminomethyl-4-oxo-3,4-dihydro-quinazoline-5-carboxylic acid methylester, 67. To a solution of 25 mL of 7N NH₃ (large excess) in MeOH at 0°C. was added compound 4 (1.0 g, 4.0 mmol) in a sealed tube. The mixturewas then heated to 60° C. for 4 hours. The mixture was evaporated todryness, dissolved and re-evaporated in 2×50 mL of CH₂Cl₂. Product wasused as is without further purification.

Procedure I to prepare2-(tert-butoxycarbonylamino-methyl)-4-oxo-3,4-dihydro-quinazoline-5-carboxylicacid methyl ester, 68. To a solution of 50 mL CH₂Cl₂ of with 2 mL of TEA(excess), catalytic DMAP and compound 67 (from Procedure H) was addedboc anhydride (2.6 g, 3 eq) at room temperature. Reaction was allowed tostir for 60 minutes, during which time all solids went into solution.The solution was evaporated to dryness and purified via columnchromatography using CH₂Cl₂ and 5% MeOH to afford 0.5 g of analyticallypure compound, 68.

Procedure J to prepare 69.5 g of compound 68 was dissolved in 10 mL ofhydrazine monohydrate and 25 mL of ethanol. The mixture was refluxed forfour hours until no starting material was detected by TLC. Reaction wascooled, poured over 100 mL of cold water and extracted with 2×25 mL ofEtOAc. Organic layers were dried with brine and then magnesium sulfate.Purified via column chromatography using 9:1 CH₂Cl₂/MeOH to afford 2.7 gof analytically pure compound 69. MS (ES−): 314; ¹H NMR (400 MHz,CDCl₃): 1.45 (s, 9H), 3.90 (s, 2H), 6.15 (bs, 1H), 6.94-7.30 (m, 3H),12.38-12.43 (m, br, 2H). Anal. Calcd. for C₁₅H₁₇N₅O₃.0.2 H₂O: C, 56.49;H, 5.50; N, 21.96; Found: C, 56.61; H, 5.60; N, 21.85.

Preparation of8-aminomethyl-2,9-dihydro-1,2,7,9-tetraaza-phenalen-3-one, 70

Procedure K to prepare 70. 250 mg of compound 69 was dissolved in 10 mLof CH₂Cl₂ along with 4 mL of TFA. The reaction was allowed to stir atroom temperature overnight resulting in a heavy white precipitate, whichwas filtered off and washed with CH₂Cl₂ and dried under vacuum to afforda quantitative yield of analytically pure material, a TFA salt ofcompound 70. MS (ES+): 216; ¹H NMR (400 MHz, D₂O): 3.97 (s, 2H), 6.91(d, J=8.2 Hz, 1H), 7.23 (d, J=7.8 Hz, 1H), 7.43 (t, J=8.0 Hz, 1H). Anal.Calcd. for C₁₀H₉N₅O. 1.3 CF₃COOH. 0.2 H₂O: C, 41.27; H, 2.86; N, 19.10;Found: C, 41.00; H, 3.04; N, 19.25.

Preparation of4-methyl-N-(3-oxo-2,9-dihydro-3H-1,2,7,9-tetraaza-phenalen-8-ylmethyl)-benzenesulfonamide,72

Synthesized using 4-methylbenzene sulfonyl chloride and compound 70 forGeneral Procedure L. 20% yield for compound 72. MS (ES−): 368; ¹H NMR(400 MHz, CDCl₃): 2.27 (s, 3H), 3.93 (s, 2H), 7.28-7.43 (m, 3H),7.67-7.78 (m, 4H), 11.43 (s, 1H), 11.83 (s, 1H). Anal. Calcd. forC₁₇H₁₅N₅O₃S: C, 55.27; H, 4.09; N, 18.96; S, 8.68; Found: C, 54.93; H,4.09; N, 18.63; S, 8.33.

Preparation ofN-(3-oxo-2,9-dihydro-3H-1,2,7,9-tetraaza-phenalen-8-ylmethyl)-benzenesulfonamide,74

Synthesized using benzene sulfonyl chloride and compound 70 for GeneralProcedure L. 25% yield for compound 74. MS (ES−): 354; ¹H NMR (400 MHz,CDCl₃): 3.95 (d, J=5.0 Hz, 2H), 7.44 (d, J=7.8 Hz, 1H), 7.45-7.60 (m,3H), 7.67-7.77 (m, 2H), 7.91-7.93 (m, 2H), 8.24 (t, J=8.8 Hz, 1H), 11.24(s, 1H), 11.83 (s, 1H). Anal. Calcd. for C₁₆H₁₃N₅O₃S. 1.0 H₂O: C, 51.47;H, 4.05; N, 18.76; S, 8.59; Found: C, 51.17; H, 4.20; N, 18.73; S, 8.31.

Preparation ofN-(3-oxo-2,9-dihydro-3H-1,2,7,9-tetraaza-phenalen-8-ylmethyl)-acetamide,75

Synthesized using acetic anhydride and compound 70 for General ProcedureL. 22% yield for compound 75. MS (ES−): 256; ¹H NMR (400 MHz, DMSO-d₆):1.91 (s, 3H), 4.05 (s, 2H), 7.32 (d, J=8.1 Hz, 1H), 7.49-7.81 (m, 2H),8.40 (t, J=8.3 Hz, 1H). 11.25 (s, 1H), 11.75 (s, 1H). Anal. Calcd. forC₁₂H₁₁N₅O₂.0.5 H₂O: C, 54.13; H, 4.54; N, 26.30; Found: C, 54.14; H,4.52; N, 26.00.

Preparation of4-nitro-N-(3-oxo-2,9-dihydro-3H-1,2,7,9-tetraaza-phenalen-8-ylmethyl)-benzamide,76

Synthesized using 4-nitro-benzoyl chloride and compound 70 for GeneralProcedure L. 25% yield for compound 76. MS (ES−): 363; ¹H NMR (400 MHz,CDCl₃): 3.99 (s, 2H), 7.18-7.20 (m, 1H), 7.34-7.38 (m, 1H), 7.75-7.90(m, 2H), 8.20-8.32 (m, 2H), 8.40-8.48 (m, 3H).

In vitro PARP Inhibitory Potency—IC₅₀

A convenient method to determine IC₅₀ of a PARP inhibitor compound is aPARP assay using purified recombinant human PARP from Trevigan(Gaithersburg, Md.), as follows: The PARP enzyme assay is set up on icein a volume of 100 microliters consisting of 100 mM Tris-HCl (pH 8.0), 1mM MgCl₂, 28 mM KCl, 28 mM NaCl, 3.0 μg/ml of DNase I-activated herringsperm DNA (Sigma, Mo.), 30 micromolar [³H]nicotinamide adeninedinucleotide (62.5 mci/mmole), 15 micrograms/ml PARP enzyme, and variousconcentrations of the compounds to be tested. The reaction is initiatedby adding enzyme and incubating the mixture at 25° C. After 2 minutes ofincubation, the reaction is terminated by adding 500 microliters of icecold 30% (w/v) trichloroacetic acid. The precipitate formed istransferred onto a glass fiber filter (Packard Unifilter-GF/C) andwashed three times with 70% ethanol. After the filter is dried, theradioactivity is determined by scintillation counting. The compounds ofthis invention were found to have potent enzymatic activity in the rangeof a few nanomolar to 20 micromolar in IC₅₀ in this inhibition assay.

Using the PARP assay described above, approximate IC50 values wereobtained for the following compounds:

TABLE I Compound Structure IC50 nM 7

35 8

23 9

35 10

19 11

6 12

9 13

12 14

18 15

32 16

21 17

20 18

17 19

18 20

35 21

n/a 22

35 23

39 24

51 25

26 26

41 27

43 28

29 30

13 31

28 32

31 33

n/a 34

49 35

44 36

19 37

12 38

20 39

15 40

39 41

42 42

13 43

38 44

21 45

49 46

11 50

52 51

15 52

21 53

23 54

14 55

18 56

27 57

17 58

13 59

23 60

24 61

27 62

22 63

19 64

15 65

22 66

45 69

47 72

171 74

23 75

30 76

10Efficacy In Vivo for Compound 131) Mouse Intracranial Model of B16 Melanoma:

The murine melanoma cell line B16 of C57BL/6J (H-2^(b)/H-2^(b)) originwas cultured in RPMI-1640 containing 10% fetal calf serum (Invitrogen,Milan, Italy), 2 mM L-glutamine, 100 units/ml penicillin and 100 μg/mlstreptomycin (Flow Laboratories, Mc Lean, Va.), at 37° C. in a 5% CO₂humidified atmosphere. TMZ was provided by Schering-Plough ResearchInstitute (Kenilworth, N.J.). Compound 13 was dissolved in 70 mM PBSwithout potassium.

For intracranial transplantation, cells (10⁴ in 0.03 ml of RPMI-1640)were injected intracranially (ic) through the center-middle area of thefrontal bone to a 2 mm depth, using a 0.1 ml glass microsyringe and a27-gauge disposable needle. Murine melanoma B16 cells (10⁴) wereinjected ic into male B6D2F1 (C57BL/6×DBA/2) mice. Before tumorchallenge, animals were anesthetized with ketamine (100 mg/kg) andxylazine (5 mg/kg) in 0.9% NaCl solution (10 ml/kg/ip). Histologicalevaluation of tumor growth in the brain was performed 1-5 days aftertumor challenge, in order to determine the timing of treatment.

The compound 13 was administered per os 15 min before TMZ. Control micewere always injected with drug vehicles. In tumor-bearing mice treatmentstarted 48 h after challenge, when tumor infiltration in the surroundingbrain tissue was histologically evident. Mice were treated with compound13 by oral gavage once a day for five days, at the doses of 10 mg/kg.

In tumor-bearing mice, treatment started on day 2 after challenge, whentumor infiltration in the surrounding brain tissue was histologicallyevident. Mice were treated daily with compound 13 plus TMZ for 5 daysand monitored for mortality for 90 days. Median survival times (MST)were determined and the percentage of increase in lifespan (ILS) wascalculated as: {[MST (days) of treated mice/MST (days) of controlmice]−1}×100. Efficacy of treatments was evaluated by comparing survivalcurves between treated and control groups.

All procedures involving mice and care were performed in compliance withnational and international guidelines (European Economy CommunityCouncil Directive 86/109, OLJ318, Dec. 1, 1987 and NIH Guide for careand use of laboratory animals, 1985).

Survival curves were generated by Kaplan-Meier product-limit estimateand statistical differences between the various groups (8 animals/group)were evaluated by log-rank analysis with Yates correction (softwarePrimer of Biostatistics, McGraw-Hill, New York, N.Y.). Statisticalsignificance was determined at a p=0.05 level. Differences wereconsidered statistically significant when P<0.05.

The results indicate oral administration of 10 mg/kg compound 13significantly increased the survival time of mice treated with compound13+TMZ combination and was significantly higher than that observed inanimals receiving TMZ as single agent (P<0.0001). No significantdifferences in survival times were observed between control and TMZtreated groups (FIG. 1).

2) Intracranial Xenograft Model of SJGBM2 Glioma in Mice:

The compound 13 was tested in the intracranial xenograft model of SJGBM2glioma in mice (Tentori, et al. Clin. Cancer Reser. 2003, 9, 5370). Forthis purpose compound 13 was given once at 15 min pre-TMZ at 10 mg/kg,po.

A dose of 10 mg/kg compound 13 was found to be efficacious (FIG. 2). Itscombination with TMZ increased MTS from 22.5 d (TMZ alone) to 25 d(P=0.002).

Efficacy In Vivo for Compound 37

1) Mouse Intracranial Model of B16 Melanoma:

The experiment was performed as described above for Compound 13. It wasinvestigated whether oral administration of Compound 37 (5 mg/kg or 12.5mg/kg), might increase the efficacy of TMZ against B16 melanoma growingat the CNS site. In mice bearing B16 melanoma, the results indicatedthat the mean survival time of the groups treated with Compound 3712.5mg/kg+TMZ combination was significantly higher than that observed inanimals receiving TMZ as single agent (FIG. 3).

2) Intracranial Xenograft Model of SJGBM2 Glioma in Mice:

The efficacy of Compound 37 was then investigated using an orthotopicmodel of a human glioblastoma multiforme xenograft (SJGBM2) in nudemice. The response of SJGBM2 to TMZ, used as single agent or incombination with Compound 37 (10 mg/kg or 20 mg/kg) for five days or incombination with Compound 37 (MGI25036) 10 mg/kg for five days followedby a 14-day treatment with Compound 37100 mg/kg as single agent, isshown in FIG. 4. The results indicate that oral administration ofCompound 37 (10 mg/kg or 20 mg/kg)+TMZ significantly prolonged survivalof tumor bearing mice with respect to controls or to animals treatedwith TMZ. It should be noted that in this tumor model TMZ wasineffective. Treatment with 10 mg/kg Compound 37+TMZ for five daysfollowed by a high dose of Compound 37 (100 mg/kg) for 14 dayssignificantly increased animal survival with respect to 10 mg/kgCompound 37+TMZ for five days.

3) Enhancement of Radiation Treatment of Head and Neck Squamous CellCarcinoma

Human HNSCC cell line JHU012 was used, having been previouslygenetically characterized and originally derived at the Johns HopkinsUniversity Head and Neck Laboratories from human tumor explants. Thecell line was maintained in RPMI 1640 medium with 10% fetal bovine serumand 1% penicillin/streptomycin at 5% CO₂ in 37° C. humidifiedincubators. Experiments were performed on 6-week-old male BALB/c nudemice nu/nu. The animals were randomly divided into the followingtreatment groups: Group 1—controls, Group 2—Radiation alone (2 Gray(gy)/day for 2 days), Group 3—100 mg/kg Compound 37 alone orally (PO)qdx17, Group 4—30 mg/kg Compound 37 PO+Radiation, Group 5—100 mg/kgCompound 37 PO+Radiation, with each group consisting of 8 mice. Micewere anesthetized by intraperitoneal injection of 3-5 mLtribromoethanol. Tumors were established at the right flank bysubcutaneous injection of 1×10⁷ cells. Fourteen days post cell injectiontumors were surgically exposed and measured in 3 dimensions usingcalipers. Compound 37 was then dosed orally in treatment Groups 3-5. InGroups 4 and 5, animals received Compound 3715 minutes prior toradiation (2 gy/day for 2 days). At day 31 post tumor cell inoculation,tumors were again surgically exposed and measured in 3 dimensions usingcalipers A significant inhibition of tumor growth was observed in Group5 treated with 100 mg/kg orally administered Compound 37+Radiation(tumor volume at end of experiment=209.04 mm³) compared to the controlGroup 1 (tumor volume at end of experiment=585.9 mm³ p<0.01). (FIG. 5)Compound 37 at 30 mg/kg in combination with radiation had no significanteffect on tumor growth inhibition compared to radiation alone (FIG. 5).In addition, 100 mg/kg Compound 37 PO qdx17 alone had no significanteffect on tumor growth inhibition compared to vehicle controls (FIG. 5).This indicates an enhanced effect when the higher dose of Compound 37was combined with radiation as opposed to either treatment modalityalone.

4) Effect of Compound 37 on Tumor Growth in Mice Bearing BRCA-1Deficient Tumors

1×10⁶ BRCA-1 null cells were injected subcutaneously on the right flankof female nu/nu mice (6-7 weeks old; Harlan Sprague Dawley, IndianapolisInd.). After approximately 10-14 days, the tumors were approximately 100mm³. Mice were sorted into groups so that mean tumor size was similaramong groups with minimum standard deviations. Dosing started the dayafter sorting and tumor volume was monitored three times per week.Tumors were measured in two diameters and volume calculated by (l×w)²/2.Mice were removed from the study when tumors reached 1500 mm³. “Time toEndpoint” or TTE (the number of days it takes for the tumor to reach1500 mm³ or greater) is the endpoint of the study. Compound 37 wasweighed out every 2-3 days and solubilized in sterile bottled water (J.T. Baker, Ultrapure Bioreagent 4221-02) to 10 mg/ml. The compound wasdosed orally, daily for 28 days from start of the study day 1. Apositive control was utilized, using a well known PARP inhibitor shownto be effective as a stand alone agent in the BRCA models (Bryant etal). The positive control agent was dosed at 25 mg/kg IP qdx5 from startof experiment. 100 mg/kg Compound 37 was effective in significantlyretarding tumor growth in the BRCA-1 null model both times tested. Whenthe dosing of Compound 37 was stopped at day 28, the tumors start togrow approximately 10-14 days later. Compound 37 not only significantlydelayed tumor growth compared to vehicle controls but also delayed tumorgrowth compared to the positive control (p<0.05) in both experiments.

A study was conducted to compare the bioavailablity and brain plasmalevels of various mammals administered with the disclosed compounds anda similar prior art compound. The prior art compound has the followingformula:

The comparative study was conducted as follows:

PARP inhibitors in water solutions were dosed either by bolus (<1 min)intravenous injection, or by oral gavage. For dogs, intravenous and oraldosing was performed in a crossover design with a one-week washoutperiod between dose routes. The screening dose was 30 mg/kg for eachcompound. For mice, three animals per time point were sacrificed by CO₂asphyxia and blood collected by cardiac puncture. For rats and dogs,serial blood samples were taken at various time points from theindicated number of animals. For rats, the volume of blood sampled wasimmediately replaced with 2× volume of 1:1 donor rat blood:heparinizedsaline. The blood samples were transferred to heparinized containers,briefly mixed, and stored on ice until centrifugation to prepare plasma.The plasma was transferred to fresh containers and stored at ≦−70° C.until bioanalysis. In some cases brains or tumor tissue were collectedafter sacrifice and stored at ≦−70° C. until bioanalysis.

Plasma samples were processed by precipitation with acetonitrile,evaporation and reconstitution. Brain and tumor tissue samples werehomogenized with phosphate buffered saline, pH 7.4, precipitated withacetonitrile, followed by evaporation and reconstitution. Thereconstituted samples were analyzed vs. matrix calibration standards byLC-MS/MS. The bioanalytical method performance was verified by theperformance of quality control samples. Generally, the plasma lowerlimit of quantitation was 5 ng/mL. Tissue lower limits of quantitationdepended on the degree of dilution during homogenization, but usuallywere 15 to 20 ng/g.

Plasma, brain, and tumor concentration data were processed bynoncompartmental pharmacokinetic analysis using WinNonlin ProfessionalVersion 4.1. AUC was calculated using the Linear/Log rule. Time pointsfor the Lambda Z phase were selected by visual inspection. The slopes ofterminal phases were calculated by unweighted linear regression.

Selective PARP inhibitors were tested for basic plasma and tissuepharmacokinetic properties in mice, rats, and dogs. After assessment,this family compounds appear to be orally bioavailable in all speciesand to penetrate brain and tumor tissue. Table 1 summarizes the oralbioavailability (PO) for compounds 8, 13, 36 and 37 and the comparativecompound in mice and rats and brain/plasma ratio (B/P) for these fivecompounds in mice and rats.

The results of the comparative study are summarized in Table II. Theresults show that while the prior art compound has good bioavailabilitythe prior art compound has a ratio of brain to plasma levels that isvery low. Unexpectedly, the disclosed compounds of Formula (I) have agood ratio of brain to plasma level compared to the prior art compound.These results show the disclosed compounds are unexpectedly available tothe central nervous system where needed for therapeutic benefit ascompared to the prior art compound.

Table II. Comparison of Bioavailabity (PO) and Ratio of Brain to Plasmalevels (B/P) for selected compounds of Formula (I) relative to a relatedprior art compound.

B/P in Compound PO in mice mice PO in rats B/P in rats

77% <5% 77% <5%

49% 49% 58% 40%

61% 46% 51% 42%

75% 30-64% 50% 71-117%

81% 26% 45% 36%

The invention being thus described, it will be obvious that the same maybe varied in many ways. Such variations are not to be regarded as adeparture from the spirit and scope of the invention, and all suchmodifications are intended to be included within the scope of thefollowing claims.

INCORPORATION BY REFERENCE

All publications, patents, and pre-grant patent application publicationscited in this specification are herein incorporated by reference, andfor any and all purposes, as if each individual publication or patentapplication were specifically and individually indicated to beincorporated by reference. In the case of inconsistencies the presentinvention will prevail.

1. The compound 37

or a pharmaceutically acceptable salt or ester thereof.
 2. A compound ofclaim 1, wherein the compound is a pharmaceutically acceptable salt orester of compound
 37. 3. A compound of claim 1, wherein the compound isa pharmaceutically acceptable salt of compound
 37. 4. A compound ofclaim 3, wherein the pharmaceutically acceptable salt is the salt of anorganic acid.
 5. The compound


6. A pharmaceutical composition comprising the compound of claim 1, anda pharmaceutically acceptable carrier.
 7. A pharmaceutical compositioncomprising the compound of claim 2, and a pharmaceutically acceptablecarrier.
 8. A pharmaceutical composition comprising the compound ofclaim 3, and a pharmaceutically acceptable carrier.
 9. A pharmaceuticalcomposition comprising the compound of claim 4, and a pharmaceuticallyacceptable carrier.
 10. A pharmaceutical composition comprising thecompound of claim 5, and a pharmaceutically acceptable carrier.
 11. Thepharmaceutical composition of claim 6, further comprising achemotherapeutic agent selected from the group consisting oftemozolomide, adriamycin, camptothecin, carboplatin, cisplatin,daunorubicin, docetaxel, doxorubicin, interferon-alpha, interferon-beta,interferon-gamma, interleukin 2, irinotecan, paclitaxel, topotecan, ataxoid, dactinomycin, danorubicin, 4′-deoxydoxorubicin, bleomycin,pilcamycin, mitomycin, neomycin, gentamycin, etoposide, 4-OHcyclophosphamide, a platinum coordination complex, and mixtures thereof.12. The pharmaceutical composition of claim 11, wherein saidchemotherapeutic agent is temozolomide, or a salt thereof.